JP5059412B2 - Alicyclobacillus sp. Polypeptide - Google Patents

Description

Field of Invention:
The present invention relates to functional and operational polypeptides encoded by polynucleotides encompassed by the genome of Alicyclobacillus sp. Deposited under accession number DSM15716. The present invention further relates to said polynucleotides encoding such polypeptides or promoting their expression, structures of such polynucleotides, and methods for preparing said polypeptides. Furthermore, the present invention relates to a composition comprising said polypeptide and polynucleotide, and the use of said polypeptide. Furthermore, the present invention relates to a bacterium Alicyclobacillus sp. As deposited under accession number DSM15716.

Background of the invention:
Several enzymes from the genus Alicyclobacillus described in the following literature are known: Matzke et al .; Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius ATCC 2700; reclassification of a group of enzymes; Submitted (MAR-1999) to the EMBL / GenBank / DDBJ databases or Koivula .; Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius. J. Gen. Microbiol. 139: 2399 (1993 ; Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modeling; Biochem. J. 328: 277 (1997) or Tsuruoka etc.; Collagenolytic Serine- Carboxyl Proteinase from Alicyclobacillus sendainensis Strain NTAP- 1: Purification, Charac-terization, Gene Cloning, and Heterologous Expression; Submitted (MAY-2002) to the EMBL / GenBank / DDBJ databases; Eckert K. & Schneider E., A thermoacidophilic endoglucanase (ceIB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofu- ranosidases belonging to family 51 of glycosyl hydrolases; Eur. J. Biochem., 270: 3593-3602, 2003.

In the study of new enzymes, it is also known to screen for such new enzymes by deferring potential candidates for a particular enzyme assay. This approach is limited to the effectiveness of enzyme assays and does not allow the identification of functional enzymes or polypeptides whose activity is still unknown.
In addition, complete genome sequencing is based on all genes from a given microorganism as described in Fleischmann et al.; Whole genome sequences and assembly of Haemophilus influenzae Rd; Nature 269: 496-512; (1995) It is a known method for obtaining information.

  Most enzymes for industrial use are enzymes that are secreted into the medium by microorganisms. However, only a few percent of microbial genomes encode proteins that are secreted. For example, only about 4% of the Bacillus subtilis genome or its closely related protein encodes a secreted protein (Van Dijl et al .: Protein transport pathways in Bacillus subtilis: a genome-based road map; in "Bacillus subtilis p. 337-355; AL Sonenshein (ed.); ASM Press 2002)).

One drawback of genomic sequencing is that a vast portion of the resulting sequence encodes a non-secreted protein.
Signal trapping, a method for identifying genes encompassing nucleotides encoding signal peptides using translational fusion to an extracellular reporter gene lacking its own signal, is also known (WO01 / 77315 issue).

Summary of invention:
The inventor has expressed the Alicyclobacillus strain, ie, the Alicyclobacillus sp. DSM 15716, which grows at low pH (about 4-5) and high concentration (50-60 ° C.). This strain is interesting because the polygene distance between the publicly known strain and the DSM15716 strain is significant, and the growth conditions are similar to those for some applications for industrial enzymes Is.

  The microbial genome contains thousands of different genes; some encode polypeptides and some encode RNA. Only a limited number of genomes in the genome of a microorganism encode functional polypeptides that are secreted by the microorganism into the surrounding medium that serves an external purpose for the microorganism. Such polypeptides are of industrial interest in that they can be produced in substantial amounts in a continuous process without destroying the cells that produce the polypeptide.

  Identifying and supplying polypeptides secreted from Alicyclobacillus sp. Deposited under accession number DSM15716 with functional purpose for Alicyclobacillus sp. Not only can they be used for commercial purposes, but they are the object of the present invention as they can be produced in industrially suitable processes and quantities.

  In a first aspect, the invention is at least 90% identical to the corresponding secreted polypeptide obtained from the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716 and the same of said polypeptide An isolated mature functional polypeptide that exhibits function is provided.

  In a further aspect, the present invention provides a bacterial glutamate peptidase (EC3.4.23.19).

  In a further aspect, the invention provides a polynucleotide encoding a polypeptide of the invention; a polynucleotide encoding a polypeptide operably linked to one or more regulatory sequences that direct the production of said polypeptide in a host cell A recombinant expression vector comprising the nucleotide structure of the invention; and a recombinant host cell comprising the nucleotide structure of the invention.

In a further aspect, the present invention provides:
(A) culturing a strain comprising a nucleotide sequence encoding a polypeptide of the present invention capable of expressing and secreting the polypeptide of the present invention; and (b) recovering said polypeptide. A process for the preparation of a polypeptide of the invention, comprising:
In a further aspect, the present invention provides a composition comprising a polynucleotide of the present invention, and a method for preparing such a composition comprising mixing the polynucleotide of the present invention and an excipient. To do.

In a further aspect, there is provided the use of a polypeptide of the invention or a composition comprising said polypeptide for various uses of the invention.
In a further aspect, the present invention relates to the bacterium Alicyclobacillus sp. As deposited under accession number DSM15716.
In a final aspect, the present invention provides an electronic storage medium containing information on the amino acid sequence of the polypeptide of the present invention or the nucleotide sequence of the polynucleotide of the present invention.

Array enumeration:
This application contains information in the form of a sequence listing attached to this application and also provided on the data carrier that accompanies this application. The contents of the data carrier are incorporated herein by reference. The region of SEQ ID NOs: 1-25 that encodes the mature polypeptide encodes the mature polypeptide of SEQ ID NOs: 26-50. Thus, the region of SEQ ID NO: 1 that encodes the mature polypeptide encodes the mature polypeptide encompassed by SEQ ID NO: 26, and the region of SEQ ID NO: 2 that encodes the mature polypeptide comprises the mature region encompassed by SEQ ID NO: 27 Encodes a polypeptide, and so on.

Specific description of the invention:
Definition:
The term “identity” as used herein is to be understood as a homology between two amino acids or between two nucleotide sequences. For the purposes of the present invention, the degree of identity between two amino acid sequences is determined by using AlignX (Informax inc., 7600 Wisconsin Avenue, Suite # 1100, Bethesda, MD 20814, USA) in the program of Vector NTI ver. 7.1. Determined by using. Amino acid alignments were created using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680,1994).

  The following additional parameters were used: 10 gap opening penalty, 0.05 gap extension penalty, 8 gap separation penalty range. The pairwise alignment parameters were as follows: Ktuple = 1, gap penalty = 3, gap length opening penalty = 10, gap extension penalty = 0.1, window size = 5 and diagonal = 5. Between two nucleotide sequences The degree of identity was determined using the same algorithm and software package as above, with the following settings: 10 gap penalties and 10 gap length penalties. The pairwise alignment parameters are as follows: Ktuple = 3 and window = 20.

  The term “functional polypeptide” as used herein refers to a polypeptide that constitutes an operational unit that can be expressed and secreted by a cell and that can act according to a function that is intended to be realized by the cell. Means peptide. Optionally, cofactors are required for polypeptides that adopt the intended function. One example of a functional polypeptide is a catalytically active polypeptide or enzyme that assists in cell catalysis in the environment surrounding the cell. Another example is a polypeptide that acts as a signal substance. Further examples are polypeptides that act as sensors (receptors) for environmental parameters (chemicals in the environment surrounding the cell), polypeptides that are active against other organisms (antimicrobial (poly) peptides) Or a polypeptide that contributes to the structural integrity of the cell.

The term “mature region” as used herein with respect to an amino acid sequence or portion of a polypeptide refers to an amino acid sequence or portion of a mature functional polypeptide, or a portion, region, or domain or fragment of a polypeptide. means.
The term “region of the nucleotide sequence encoding the mature polypeptide” as used herein refers to the last encoding the last amino acid of the mature polypeptide from the triplet encoding the first amino acid of the mature polypeptide. It refers to the region of the nucleotide sequence that counts up to a triplet.

  The term “secreted polypeptide” as used herein is transported to and released into the surrounding extracellular medium after expression in the cell, or at least a portion of the polypeptide is in the surroundings. Should be understood as a polypeptide bound / embedded to the cell membrane so that it is exposed to the extracellular medium.

Polypeptide of the present invention:
The present invention relates to polypeptides similar to those secreted polypeptides obtained from the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716. In particular, the present invention provides an isolation that is at least 90% identical to the corresponding secreted polypeptide obtained from the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716 and exhibits the same function of said polypeptide. Related mature functional polypeptides.

Moreover, surprisingly, the glutamate peptidase of SEQ ID NO: 27 expressed by Alicyclobacillus sp. DSM15716 is the first glutamate peptidase isolated from bacteria. Accordingly, the present invention also provides a bacterial glutamate peptidase (EC 3.4.23.19).
The polypeptides of the invention are secreted by Alicyclobacillus sp. DSM15716, particularly with the provision of functions for that particular cell.

  Among the thousands of possible genes in the genome of Alicyclobacillus sp. DSM15716, the polynucleotide of this genome was determined to be functional and translated into a functional polypeptide by the selected host cell. Encoded 25 secreted functional mature polypeptides encompassed by SEQ ID NOs: 26-50.

  Thus, Alicyclobacillus sp. DSM15716 expresses and secretes a functional mature polypeptide encompassed by SEQ ID NOs: 26-50 and, in the genome of that particular strain, SEQ ID NO: encodes the mature polypeptide. The region from 1 to 25 is a gene encoding the mature polypeptide encompassed by SEQ ID NOs: 26-50. Further, in certain embodiments, all of the genes encoding mature polypeptides encompassed by SEQ ID NOs: 26-50 are expressed, and their corresponding mature polypeptides of SEQ ID NOs: 1-25 encoding mature polypeptides. It can be secreted when culturing E. coli hosts transformed with a polynucleotide comprising these regions. By comparing the homology or identity of 25 polypeptide sequences to a known sequence, the specific function of the polypeptide is annotated. It has been determined that at least 15 of the 25 secreted functional polypeptides are enzymes.

In particular, an isolated polypeptide is
(A) a polypeptide comprising an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of mature polypeptides encompassed by SEQ ID NOs: 26-50;
(B) (i) a complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding the mature polypeptide;
(Ii) a polynucleotide probe selected from the group consisting of a complementary strand to a cDNA sequence contained in a nucleotide sequence selected from the group of SEQ ID NOs: 1 to 25 encoding a mature polypeptide, and under high stringency conditions Selected from the group consisting of polypeptides encoded by nucleotide sequences that hybridize with, wherein said polypeptide has the function of its corresponding mature polypeptide encompassed by SEQ ID NOs: 26-50.

  In one particular embodiment, the polypeptide of the invention is selected from among the enzymes secreted by the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716 and isolated by the inventor, i. Glucanase, acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease, HtrA-like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N-acetyl It is an enzyme group consisting of glucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase, phytase, phospholipase C, polysaccharide deacetylase, xylan deacetylase and sulfite oxidase.

The present invention also provides
(A) Acidic endoglucanase or acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease or HtrA-like serine secreted from the strain of Alicyclobacillus sp. Deposited as DSM accession number 15716 Protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N-acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C, polysaccharide deacetylase or xylan deacetylase and sulfite An enzyme comprising an amino acid sequence having at least 90% identity with the amino acid sequence of a mature enzyme selected from the group consisting of oxidases;

  (B) (i) Acidic endoglucanase or acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease or HtrA secreted from the strain of Alicyclobacillus sp. Deposited under DSM accession number 15716 -Like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N-acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C, polysaccharide deacetylase or xylan deacetylase A complementary strand to the nucleotide sequence encompassed by said strain encoding a mature enzyme selected from the group consisting of ase and sulfite oxidase;

      (Ii) Acid endoglucanase or acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease or HtrA-like serine secreted from the strain of Alicyclobacillus sp. Deposited as DSM accession number 15716 Protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N-acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C, polysaccharide deacetylase or xylan deacetylase and sulfite A polynucleotide selected from the group consisting of a complementary strand to a cDNA sequence contained in a nucleotide sequence encompassed by said strain encoding a mature enzyme selected from the group consisting of oxidases And Dopurobu, selected from the group consisting of polypeptides encoded by nucleotide sequence which hybridizes at high stringency conditions,

  Acid endoglucanase or acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease or HtrA-like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N Provided is an isolated enzyme having a function selected from acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C, polysaccharide deacetylase or xylan deacetylase and sulfite oxidase.

In certain embodiments, the enzyme is
(A) an enzyme having an amino acid sequence having at least 90% identity with an amino acid sequence selected from mature enzymes encompassed by SEQ ID NOs: 26-40;
(B) (i) a complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-15 encoding the mature enzyme;
(Ii) a polynucleotide probe selected from the group consisting of a complementary strand to a cDNA sequence contained in a nucleotide sequence selected from the group of SEQ ID NOs: 1-15 encoding a mature polypeptide, and under high stringency conditions An isolated enzyme selected from the group consisting of enzymes encoding nucleotide sequences that hybridize with, wherein said enzyme has the function of its corresponding mature polypeptide encompassed by SEQ ID NOs: 26-40 .

  The polypeptide of the present invention is an isolated polypeptide, preferably a preparation of the polypeptide of the present invention comprises at most 90% by weight of other naturally relevant polypeptide material (lower weight%). For example, at most 80%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, at most 10% At most 9%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, Other polypeptide materials of at most 1% by weight and at most 0.5% by weight are preferred).

  Accordingly, it is preferred that the isolated polypeptide of the present invention is at least 92% pure, i.e., the polypeptide of the present invention preferably comprises at least 92 weight of the total polypeptide material present in the preparation, And higher percentages, such as at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at most 99.5% purity are preferred, particularly preferably the polypeptides of the invention Exists in “substantially pure form”, ie the polypeptide preparation is substantially free of other polypeptide materials to which it is naturally associated. This is accomplished, for example, by preparing the polypeptides of the present invention by well-known recombinant methods.

The polypeptides of the present invention may be synthetically produced, live in nature, or a combination thereof. In certain embodiments, the polypeptides of the present invention are obtained from a microorganism, such as a prokaryotic cell, archaeal cell, or eukaryotic cell. The cells have been further modified by genetic engineering.
In certain embodiments, the polypeptides of the invention are enzymes that exhibit optimal enzyme activity at temperatures in the range of about 10 ° C to about 80 ° C, particularly about 20 ° C to about 60 ° C.

In a particular embodiment, the polypeptide of the invention is an enzyme that is functionally stable at temperatures up to 100 ° C., in particular up to 80 ° C., more particularly up to 60 ° C.
In a particular embodiment, the polypeptide of the present invention is at least 20%, in particular at least 40%, such as at least 50%, in particular at least 60%, such as at least 20% of an enzyme selected from the mature enzymes encompassed by An enzyme that exhibits an enzyme activity of at least 70%, more particularly at least 80%, such as at least 90%, most particularly at least 95%, such as about or at least 100%.

  In particular, the isolated mature functional polypeptide is at least 90% identical to the corresponding secreted polypeptide obtained from the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716, and said poly An amino acid sequence exhibiting the same function of a peptide, and in particular, a polypeptide of the invention having at least 90% identity with a polypeptide sequence selected from the group consisting of the mature polypeptides encompassed by SEQ ID NOs: 26-50 Comprising, comprising or consisting of. Percent identity is in particular at least 95%, such as at least 96%, such as at least 97%, and even more particularly at least 98%, such as at least 99% or even 100%.

In another particular embodiment, the percent identity is at least 50%, in particular at least 60%, in particular at least 65%, in particular at least 70%, in particular at least 75%, in particular at least 80%, and even more particularly at least 85% identity.
In certain embodiments, the amino acid sequence of a polypeptide of the invention is no more than 10 amino acids (eg, 10 amino acids), in particular at most 5 from the mature polypeptide encompassed by SEQ ID NOs: 26-50. 1 amino acid (eg 5 amino acids), for example at most 4 amino acids (eg 4 amino acids), for example at most 3 amino acids (eg 3 amino acids), in particular at most 2 Different amino acids (eg 2 amino acids), eg 1 amino acid.

  The polypeptide of the present invention is a wild type polypeptide isolated from a natural source, for example, strain Alicyclobacillus sp. DSM15716, or other wild type strain, however, the present invention also includes a polypeptide of the present invention. Are artificially mutated by adding, substituting and / or deleting one or more amino acids from said polypeptide while maintaining the function and / or other properties of the polypeptide Includes the body. Thus, a polypeptide of the invention comprises an amino acid sequence comprising or consisting of a mature polypeptide wherein at least one amino acid substitution, deletion and / or insertion is encompassed by SEQ ID NOs: 26-50. It may be an artificial mutant that has been performed.

  The polypeptides of the present invention also include functional fragments of the amino acid sequences described herein, and functional fragments of nucleic acids that encode functional fragments of the amino acid sequences described herein, eg, Fragment of mature enzyme secreted from strain of bacterial Alicyclobacillus sp. Deposited as accession number DSM15716 as described, for example, acid secreted from strain of bacterial Alicyclobacillus sp. Deposited as accession number DSM15716 Endoglucanase, acid cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease, HtrA-like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N- Acetylglucosaminidase, pe Chijiru - prolyl - including isomerase, acid phosphatase, phytase, phospholipase C, polysaccharide deacetylase, a fragment of an enzyme selected from the group consisting of xylan deacetylase and sulfite oxidase.

  Artificial mutants are constructed by standard techniques known in the art, usually with subsequent screening and / or characterization. Standard techniques include conventional mutagenesis, eg, UV irradiation of cells, or treatment of cells with chemical mutagens as described in the following literature: Gerhardt et al. (1994); WO In vivo gene shuffling as described in 97/07205; in vivo gene shuffling as described in Stemmer, (1994) or WO 95/17413; described by Eisenstadt E. et al., (1994) Random mutagenesis such as: PCR techniques as described by Poulsen et al. (1991); families as described by JE Ness, et al., Nature Biotechnology, vol. 17, pp. 893-896 (1999) Shuffling; Sambrook et al. (1989), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY. Specific site mutagenesis, Ford et al., 1991, Protein Expression and Purification 2, p. 95-107 General described.

  Such standard genetic engineering methods also encode one or more parent enzymes of the invention, express the enzyme variant in a suitable host cell, and select a preferred variant from the nucleotide sequence of the variant nucleotide sequence. Can be used to prepare diversified libraries. A diversified library can be established by a wide range of techniques known in the art (Reetz MT; Jaeger KE, in Biocatalysis-from Discovery to Application edited by Fessner WD, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p. 389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano etc. , Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998).

  In certain aspects of the invention, amino acid alterations (in artificial mutants and wild type enzymes) are of minor nature, ie, conservative amino acid substitutions that do not significantly affect protein folding and / or activity; Small deletions typically from 1 to about 30 amino acids; small amino- or carboxy-terminal-, eg, amino-terminal methionine residue extensions; small linker peptides of up to about 20-25 residues Or a small extension that facilitates purification by altering the net charge or other function, such as the poly-histidine system, antigenic epitopes or binding domains.

  Examples of conservative substitutions are basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic Within the group of amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, and threonine). In general, amino acid substitutions that do not alter inactivity and / or impair protein function are known in the art and described, for example, by H. Neurath and RL Hill, 1979, The Proteins, Academic Press, New York Has been. The most common exchanges are: Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe , Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly and vice versa.

In certain embodiments, the amino acid alteration is of such a property that the physiochemical properties of the polypeptide are altered. For example, amino acid changes can be made that improve the thermal stability of the polypeptide, change substrate specificity, change pH optimization, and the like.
In particular, such substitutions, deletions in the polypeptides of the invention, in particular those polypeptides selected from the group consisting of the mature polypeptides encompassed by SEQ ID NOs: 26-50 to generate artificial variants, And / or the number of insertions is at most 10, such as at most 9, such as at most 8, such as at most 7, such as at most 6, such as at most 5, most preferably at most 4, such as at most 3, such as At most 2, especially at most 1.

  In a particular embodiment, the artificial variant is a variant that has altered, preferably reduced, immunogenicity, especially allergenicity, in animals, such as humans, compared to the true enzyme. As used herein, the term “immunogenic” refers to an artificial variant that elicits an altered, particularly reduced immune response when administered intravenously, cutaneously, subcutaneously, orally and intratracheally in an animal. It should be understood as an ability. As used herein, the term “immune response” means that administration of an artificial mutant causes a change in the level of immunoglobulins, eg, IgE, IgG and IgM in the animal body, or a change in cytokine levels in the animal body. To do.

  Methods for preparing variants with altered immunogenicity, mapping protein immunogenic / antigenic epitopes, and measuring immune responses are well known in the art, And it is described in WO 92/10755, WO 00/26230, WO 00/26354 and WO 01/31989. As used herein, the term “allergenic” should be understood as the ability of an artificial mutant to induce altered, particularly reduced production of IgE in an animal, and the ability to bind IgE from said animal. is there. The allergenicity resulting from intratracheal administration of polypeptide variants to animals is of particular interest (known as respiratory allergy).

In a further aspect, the polypeptide of the invention comprises
(I) a complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding the mature polypeptide;
(Ii) a complementary strand to a cDNA sequence contained in a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding the mature polypeptide;
(Iii) a polynucleotide probe selected from the group consisting of a fragment of (i) or (ii) that encodes a secreted polypeptide having the function of its corresponding mature polypeptide encompassed by SEQ ID NOs: 26-50; A polypeptide encoded by a nucleotide sequence that hybridizes at least under high stringency conditions (J. Sambrook, EF Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York) .

  In particular, the polypeptide of the present invention comprises a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding a mature polypeptide, or a polynucleotide comprising a sequence that differs from them due to the degeneracy of the genetic code. Coded. More specifically, the polypeptide of the present invention is a polynucleotide comprising a nucleotide sequence selected from the group of SEQ ID NOs: 1 to 25 encoding a mature polypeptide, or a sequence that differs from them due to the degeneracy of the genetic code Coded by

  The nucleotide sequence of the region of SEQ ID NO: 1-25 encoding the mature polypeptide or a subsequence thereof, and the amino acid sequence of the mature polypeptide encompassed by SEQ ID NO: 26-50 or a fragment thereof are methods well known to those skilled in the art. Can be used to design polynucleotide probes to identify and clone DNA encoding enzymes of the invention from different genus or species strains. In particular, such probes can be used for hybridization with the genome or cDNA of the genus or species of interest, according to standard Southern blot methods, to identify and isolate the corresponding gene therein. .

  Such probes should be considerably shorter than the complete sequence, but should be at least 15, preferably at least 25, more preferably at least 35, for example at least 70 nucleotides in length. Preferably, however, the nucleotide probe is at least 100 nucleotides in length. For example, a polynucleotide probe can be at least 200 nucleotides in length, at least 300 nucleotides in length, at least 400 nucleotides in length, or at least 500 nucleotides in length.

Longer probes can be used, such as polynucleotide probes that are at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probe is typically labeled with, for example, ³² P, ³ H, ³⁵ S, biotin or avidin to detect its corresponding gene.

  Thus, genomic DNA or cDNA libraries prepared from such other organisms can be screened for DNA that hybridizes to the probe and encodes the enzyme of the invention. Genomic or other DNA from such organisms can be separated by agarose or polyacryl gel electrophoresis or other separation techniques. DNA from the library or isolated DNA can be transferred or immobilized to nitrocellulose or other suitable carrier material. Identify clones or DNAs that have the required homology and / or identity, or that are homologous and / or identical to nucleotides selected from the region of SEQ ID NOs: 1-25 encoding the mature polypeptide To do so, a carrier material with immobilized DNA is used for Southern blots.

  For the purposes of the present invention, hybridization will hybridize to a nucleotide sequence selected from the region of SEQ ID NO: 1-25 encoding the mature polypeptide under very low to very high stringency conditions. , Indicates hybridization to a labeled polynucleotide probe. Molecules to which the polynucleotide probe hybridizes under these conditions can be detected using X-ray film or by any other method known in the art. It is to be understood that when the term “polynucleotide probe” is used herein, such a probe comprises at least 15 nucleotides.

In an interesting embodiment, the polynucleotide probe is a complementary strand of nucleotides selected from the region of SEQ ID NOs: 1-25 encoding the mature polypeptide.
In another interesting embodiment, the polynucleotide probe is the complementary strand of a nucleotide sequence encoding an enzyme selected from the group of SEQ ID NOs: 26-50. In a further interesting embodiment, the polynucleotide probe is the complementary strand of the mature polypeptide coding region of a nucleotide sequence selected from the region of SEQ ID NOs: 1-25 encoding the mature polypeptide.

  For probes that are at least 100 nucleotides in length, high to very high stringency conditions are sheared at 5 × SSPE, 1.0% SDS, 5X Denhardt solution, 100 μg / ml according to standard Southern blot methods. And prehybridization and hybridization at 42 ° C. in denatured salmon sperm DNA. Preferably, a long probe of at least 100 nucleotides in length does not contain more than 1000 nucleotides. For a length probe of at least 100 nucleotides in length, the carrier material will ultimately be 3 times (high stringency), in particular 0.1%, using 0.1 × SSC, 0.1% SDS solution for 15 minutes each at 60 ° C. Washed 3 times (very high stringency) for 15 minutes each at 68 ° C using xSSC, 0.1% SDS solution.

Although not particularly preferred, short probes, such as probes that are about 15 to 99 nucleotides in length, such as about 15 to about 70 nucleotides in length, can also be used. For such short probes, stringent conditions are 0.9 M NaCl, 0.09 M Tris-HCl, pH 7.6, 6 mM EDTA, 0.5% NP-40, 1 × Denhardt's solution, 1 mM sodium pyrophosphate, More than the Tm calculated according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48: 1390) in 1 mM monobasic sodium phosphate, 0.1 mM ATP and 0.2 mg yeast RNA per ml. Defined as prehybridization, hybridization, and post-hybridization washing according to standard Southern blot methods at temperatures about 5 ° C. to about 10 ° C. lower.
For short probes that are about 15 to about 99 nucleotides in length, the carrier material is calculated from the Tm calculated using 6 × SSC and 0.1% SDS for 15 minutes, once, and 6 × SSC. Is also washed twice at a temperature about 5 ° C. to about 10 ° C. for 15 minutes each.

SEQ ID NO: 26 acid endoglucanase or acid cellulase :
In a particular embodiment, the polypeptide of the invention comprises an acid endoglucanase or acid cellulase, more particularly SEQ ID NO: obtained from a strain of the alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716 And at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly with the mature acid endoglucanase or acid cellulase included in Is an acidic endoglucanase or acidic cellulase comprising or consisting of an amino acid sequence having at least 99%, or most particularly 100% identity.

  More particularly, the mature acid endoglucanase or acid cellulase comprises or consists of a sequence from positions 25-959 of SEQ ID NO: 26. As used herein, acidic endoglucanase is defined as an enzyme that terminally hydrolyzes 1,4-β-D-glucoside bonds in cellulose, lichenin or cereal β-D-glucan, especially under acidic conditions. As used herein, acidic cellulase is defined as an enzyme that terminally hydrolyzes 1,4-β-D-glucoside bonds in cellulose under acidic conditions.

SEQ ID NO : 27 glutamate peptidase :
In a particular embodiment, the polypeptide of the invention is encompassed by Aricyclobacillus sp., In particular glutamate peptidase obtained from a strain of the bacterial Alicyclobacillus sp. Deposited under accession number DSM15716, more particularly SEQ ID NO: 27. Mature acid endoglucanase or acid cellulase with at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% Or, most particularly, a glutamate peptidase comprising or consisting of an amino acid sequence having 100% identity. More particularly, the mature acid glutamate peptidase comprises or consists of a sequence from positions 33-272 of SEQ ID NO: 27. As used herein, glutamate peptidase is defined as an enzyme that hydrolyzes proteins or peptides and contains the conserved active site residues Q and E.

  Glutamate peptidase (PepG) (EC3.4.23.19) was previously classified as aspartyl protease (A4), but has been reclassified by MEROPS (http://merops.sanger.ac.uk/) , Fujinaga, Cherney, Oyama, Oda & James (2004), The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scy- talidium lignicolum. Recognizes catalytic peptidases, namely glutamate peptidases. The known glutamate peptidases were previously A4 and are now all included in the family that becomes G1 (Fujinaga M, Cherney MM, Oyama H, Oda K, James MN .; The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scy-talidium lignicolum; Proc. Natl. Acad. Sci. USA; 101 (10); pp. 3364-9; Epub 01-Mar-2004; 09-Mar-2004).

  It is clear that the polypeptide of SEQ ID NO: 27 is a glutamate peptidase, consisting of a sequence of the following multiple sequences confirming that the active site residues Q and E are conserved in SEQ ID NO: 27:

SWISSPROT_P24665:
(Aspergillus niger) Aspergillus pepsin II: SEQ ID NO: 55
TREMBL_Q9P8R1:
(Sclerotinia sclerotiorum) Endopeptidase EapC: SEQ ID NO: 56
TREMBL-Q00551:
(Cryphonectria parasitica) endopeptidase EapC: SEQ ID NO: 57
SWISSPROT_P15369:
(Scytalidium lignicolum) citalid glutamic acid peptidase; SEQ ID NO: 58

TREMBL_Q00550:
(Cryphonectria parasitica) endopeptidase EapB: SEQ ID NO: 59
TREMBL_Q8X1C5:
(Talaromyces emersonii) papstatin-insensitive acid protease (fragment); SEQ ID NO: 60
SEQ ID NO: 27:
Sequence of the invention:
o = amino acid that forms the active site in Swissprot P24665
/ = Cysteine residue that forms a disulfide bond in Swissprot P24665
# = Propeptide removed from Swissprot P24665 zymogen

  Accordingly, the inventors have identified and isolated a first (G1) glutamate peptidase from cells known so far, and in particular glutamate peptidase that is active at low pH and high temperature. The closest related is a fungal G1 protease (eg, Aspergillopepsin II).

  Moreover, surprisingly, this glutamate peptidase differs from the most known fungal glutamate peptidase due to the absence of disulfide bonds in the molecule. The glutamate peptidase encompassed by SEQ ID NO: 27 contains only one cysteine and is therefore in SEQ ID NO: 55, which discloses a known fungal glutamate peptidase composed of two peptides linked by two disulfide bonds. In comparison, the protease structure does not have a disulfide bond. Thus, in the glutamic acid peptidase of Alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716, the second propeptide is deleted and, therefore, less than one maturation stage in its production Need. This is advantageous for cell generation.

SEQ ID NO : 28 or 35 polycopper oxidase :
In a particular embodiment, the polypeptide of the invention is a polycopper oxidase obtained from a strain of Alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716, more particularly SEQ ID NO: 28 or 35. At least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% Or most particularly a polycopper oxidase comprising or consisting of an amino acid sequence having 100% identity.

  More particularly, the mature polycopper oxidase comprises or consists of a sequence from positions 26-315 of SEQ ID NO: 28 or positions 50-597 of SEQ ID NO: 35. As used herein, multi-Cu-oxidase is defined as a protein having at least three spectroscopically distinct copper inclusions. Polycopper oxidase loses the ability to bind laccase, which oxidizes many different types of phenols and diamines, ascorbate oxidase, ceruloplasmin, or copper, which oxidizes so many kinds of inorganic and organic substances, and Can be part of a protein that mediates heavy metal resistance by sequestering heavy metals around bacterial cells.

SEQ ID NO: 29 or 30 serine-carboxyl protease :
In a particular embodiment, the polypeptide of the present invention comprises a serine-carboxyl protease obtained from Alicyclobacillus sp., In particular a strain of bacterial Alicyclobacillus sp. Deposited under accession number DSM15716, more particularly SEQ ID NO: 29 or 30 and at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least A serine-carboxyl protease comprising or consisting of amino acid sequences with 99%, or most particularly 100% identity.

  More particularly, the mature serine-carboxyl protease comprises or consists of a sequence from positions 190-626 of SEQ ID NO: 29 or positions 25-533 of SEQ ID NO: 30. As used herein, serine-carboxyl protease is a proteolytic enzyme fold similar to that of subtilisin with respect to the unique catalytic ternary Ser-Glu-Asp and the presence of aspartic acid residues in the oxyanion hole. Defined as a protease belonging to the enzyme type EC3.4.21.100 (pseudomonapepsin). A polypeptide sequence can be classified as a serine-carboxyl peptidase if the catalytic site amino acid is present in the sequence and if it exhibits peptide sequence similarity to the peptide sequence in MEROPS serine protease family 53.

SEQ ID NO: 31 serine protease or HtrA-like serine protease :
In a particular embodiment, the polypeptide of the present invention comprises a serine protease or HtrA-like serine protease obtained from an alicyclobacillus sp., In particular a strain of the bacterial alicyclobacillus sp. Deposited under accession number DSM15716. At least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98% with the mature serine protease or HtrA-like serine protease encompassed by SEQ ID NO: 31 More particularly a serine protease or HtrA-like serine protease comprising or consisting of an amino acid sequence having at least 99%, or most particularly 100% identity.

  More particularly, the mature serine protease or HtrA-like serine protease comprises or consists of the sequence from positions 42-411 of SEQ ID NO: 31. As used herein, a serine protease is defined as an enzyme that hydrolyzes a protein or peptide and contains a serine residue at the catalytic site. An HtrA-like protease is defined as an enzyme that degrades damaged proteins in the extracellular compartment of bacterial cells at elevated temperatures.

SEQ ID NO : 32 disulfide isomerase :
In a particular embodiment, the polypeptide of the present invention is encompassed by Alisulfobacillus sp., Particularly a disulfide isomerase obtained from a strain of the bacterial Alicyclobacillus sp. Deposited under accession number DSM15716, more particularly SEQ ID NO: 32. Mature disulfide isomerase and at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most specific Is a disulfide isomerase comprising or consisting of an amino acid sequence having 100% identity. More particularly, the mature disulfide isomerase comprises or consists of a sequence from positions 31-212 of SEQ ID NO: 32. As used herein, a disulfide isomerase is defined as an enzyme that catalyzes the translocation of intrachain and interchain sulfido bonds to form an active structure.

SEQ ID NO: 33 γ-D-glutamyl-L-diamino acid endopeptidase :
In a particular embodiment, the polypeptide of the invention comprises an alicyclobacillus sp., In particular a γ-D-glutamyl-L-diamino acid endopeptidase obtained from a strain of the bacterial alicyclobacillus sp. Deposited under accession number DSM15716, More particularly the mature γ-D-glutamyl-L-diamino acid endopeptidase encompassed by SEQ ID NO: 33 and at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97% Γ-D-glutamyl-L-diamino acid comprising, or more particularly comprising, or consisting of an amino acid sequence having at least 98%, more particularly at least 99%, or most particularly 100% identity Endopeptidase.

  More particularly, the mature γ-D-glutamyl-L-diamino acid endopeptidase comprises or consists of the sequence from positions 30-266 of SEQ ID NO: 33. As used herein, γ-D-glutamyl-L-diamino acid endopeptidase is γ-D- in L-Ala-γ-D-Glu- (L) meso-diaminopimelic acid- (L) -D-Ala. Defined as an enzyme that hydrolyzes glutamyl bonds to (L) meso-diaminopimelic acid. The omega-amino and omega-carboxyl groups of the (L) -iso-diaminopimelic acid group need not be substituted.

SEQ ID NO: 34 endo-β-N-acetylglucosaminidase :
In a particular embodiment, the polypeptide of the invention comprises an endo-β-N-acetylglucosaminidase obtained from an alicyclobacillus sp., In particular a strain of the bacterial alicyclobacillus sp. Deposited under accession number DSM15716, more particularly Mature endo-β-N-acetylglucosaminidase encompassed by SEQ ID NO: 34 and at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98% More particularly an endo-β-N-acetylglucosaminidase comprising or consisting of an amino acid sequence having at least 99%, or most particularly 100% identity.

  More particularly, mature endo-β-N-acetylglucosaminidase comprises or consists of a sequence from positions 27 to 768 of SEQ ID NO: 34. As used herein, endo-β-N-acetylglucosaminidase is a 1,4-β-linkage between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglucan heteropolymers in prokaryotic cell walls. Is defined as an enzyme that hydrolyzes.

SEQ ID NO : 36 peptidyl-proyl-isomerase :
In a particular embodiment, the polypeptide of the invention comprises a peptidyl-prolyl-isomerase, more particularly SEQ ID NO: 36, obtained from a strain of the alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession no. At least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least A peptidyl-prolyl-isomerase comprising or consisting of an amino acid sequence with 99%, or most particularly 100% identity.

  More particularly, the mature peptidyl-prolyl-isomerase comprises or consists of a sequence from positions 30 to 246 of SEQ ID NO: 36. As used herein, peptidyl-proyl-isomerase is defined as an enzyme that promotes protein folding by catalyzing the cis-trans isomerization of proline iminate peptide bonds in oligopeptides.

SEQ ID NO: 37 acid phosphatase or filterase or phospholipase C :
In a particular embodiment, the polypeptide of the present invention comprises an acid phosphatase or filterase or phospholipase C, more particularly obtained from a strain of the alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716. Mature acid phosphatase or filterase or phospholipase C encompassed by SEQ ID NO: 37 with at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98% More particularly an acid phosphatase or filterase or phospholipase C comprising or consisting of an amino acid sequence having at least 99%, or most particularly 100% identity.

  More particularly, mature acid phosphatase or filterase or phospholipase C comprises or consists of the sequence from positions 28 to 608 of SEQ ID NO: 37. Acid phosphatase is defined as an enzyme that hydrolyzes orthophosphonic acid monoesters into alcohols and phosphates. As used herein, a phytase is defined as an enzyme that removes a phosphate group from a phytate. Phospholipase C is defined as an enzyme that hydrolyzes phosphatidylcholine to 1,2-diacylglycerol and choline.

SEQ ID NO : 38 or 39 polysaccharide deacetylase :
In a particular embodiment, the polypeptide of the invention comprises a polysaccharide deacetylase or xylan deacetylase, more particularly a sequence, obtained from a strain of the alicyclobacillus sp., In particular the bacterium Alicyclobacillus sp. Deposited under accession number DSM15716. At least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98% with the mature polysaccharide deacetylase or xylan deacetylase encompassed by number 38 or 39 More particularly a polysaccharide deacetylase or xylan deacetylase comprising or consisting of an amino acid sequence having at least 99%, or most particularly 100% identity.

  More particularly, the mature polysaccharide deacetylase or xylan deacetylase comprises or consists of a sequence from positions 26-251 of SEQ ID NO: 38 or positions 22-324 of SEQ ID NO: 39. As used herein, polysaccharide deacetylase is defined as an enzyme that removes acetyl residues from a particular acetylated polysaccharide by hydrolysis. Xylan deacetylase is defined as an enzyme that removes acetyl groups from acetylated xylan.

SEQ ID NO : 40 sulfite oxidase :
In a particular embodiment, the polypeptide of the invention is encompassed by Alisulfobacillus sp., Particularly sulfite oxidase obtained from a strain of bacterial Alicyclobacillus sp. Deposited under accession number DSM15716, more particularly SEQ ID NO: 40. Mature sulfite oxidase and at least 90%, in particular at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most specific Is a sulfite oxidase comprising or consisting of an amino acid sequence having 100% identity. More particularly, the mature sulfite oxidase comprises or consists of the sequence from positions 30 to 214 of SEQ ID NO: 40. Sulfite oxidase is defined as an enzyme that oxidizes sulfite to sulfate.

SEQ ID NO : 41 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 41, more particularly SEQ ID NO: 41. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 22-257 of SEQ ID NO: 41.

SEQ ID NO : 42 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 42, more particularly SEQ ID NO: 42. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 25-1130 of SEQ ID NO: 42.

SEQ ID NO : 43 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 43, more particularly SEQ ID NO: 43. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 42 to 248 of SEQ ID NO: 43.

SEQ ID NO : 44 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, of a mature functional polypeptide encompassed by SEQ ID NO: 44, more particularly SEQ ID NO: 44. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 26-172 of SEQ ID NO: 44.

SEQ ID NO : 45 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 45, more particularly SEQ ID NO: 45. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 31-242 of SEQ ID NO: 45.

SEQ ID NO : 46 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 46, more particularly SEQ ID NO: 46. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of a sequence from positions 25-280 of SEQ ID NO: 46.

SEQ ID NO : 47 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 47, more particularly SEQ ID NO: 47. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of the sequence from positions 26-478 of SEQ ID NO: 47.

SEQ ID NO : 48 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 48, more particularly SEQ ID NO: 48. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of a sequence from positions 20-340 of SEQ ID NO: 48.

SEQ ID NO : 49 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 49, more particularly SEQ ID NO: 49. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of a sequence from positions 30-341 of SEQ ID NO: 49.

SEQ ID NO : 50 functional polypeptide :
In a particular embodiment, the polypeptide of the invention comprises at least 90%, in particular at least 95%, more particularly at least 96, a mature functional polypeptide encompassed by SEQ ID NO: 50, more particularly SEQ ID NO: 50. %, More particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly at least 100%, or a function comprising or consisting of an amino acid sequence having 100% identity Polypeptide. More particularly, the mature functional polypeptide comprises or consists of a sequence from positions 29-400 of SEQ ID NO: 50.

Polynucleotide :
The invention also relates to polynucleotides comprising, or consisting of, nucleotides encoding the polypeptides of the invention, in particular isolated polynucleotides. In certain embodiments, the nucleotide sequences are shown in SEQ ID NOs: 1-25, which include nucleotide sequences that differ from them due to the degeneracy of the genetic code. In a further embodiment, the polynucleotide of the invention comprises or consists of a nucleotide sequence selected from the region of SEQ ID NOs: 1-25 that encodes the mature polypeptide and is encompassed by SEQ ID NOs: 1-25. A modified nucleotide sequence comprising at least one modification / mutation compared to the parent nucleotide sequence to be rendered.

  Techniques used to isolate and / or clone nucleotide sequences encoding enzymes are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Includes. Cloning of the nucleotide sequences of the present invention from such genomic DNA can be accomplished using, for example, the well-known polymerase chain reaction (PCR) or expression library to detect cloned DNA fragments with shared structural features. Can be provided by using the following antibody screens. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application; Academic Press, New York. Other amplification methods such as ligase chain reaction (LCR), linked activated transcription (LAT) and nucleotide sequence based amplification (NASBA) can be used.

  The nucleotide sequence is obtained by standard cloning methods used in genetic engineering to transfer the nucleotide sequence from its natural location to a different site where it will be regenerated. Cloning methods include excision and isolation of a desired fragment comprising a nucleotide sequence encoding a polypeptide, insertion of the fragment into a vector molecule, and integration of the recombinant vector into a host cell, wherein Multiple copies or clones of the nucleotide sequence can be replicated. The nucleotide sequence can be semi-synthetic, genome of synthetic origin, cDNA, RNA, or any combination thereof.

  In particular, the polynucleotide comprises and preferably consists of a nucleotide sequence having at least 50% identity with a nucleotide sequence selected from the region of SEQ ID NOs: 1-25 encoding the mature polypeptide. In particular, the nucleotide sequence is at least 65%, more particularly at least 70%, more particularly at least 80%, more particularly more than a nucleotide sequence selected from the region of SEQ ID NO: 1-25 encoding the mature polypeptide. Is at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly 100% identity. In particular, the nucleotide sequence comprises a nucleotide sequence selected from the region of SEQ ID NO: 1-25 encoding the mature polypeptide. In a more specific embodiment, the nucleotide sequence consists of a nucleotide sequence selected from the region of SEQ ID NOs: 1-25 that encodes the mature polypeptide.

  Polynucleotides include acid endoglucanase or cellulase, aspartyl protease, polycopper oxidase, serine-carboxyl protease, serine protease or HtrA-like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo Encodes a mature enzyme selected from β-N-acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C, polysaccharide deacetylase or xylan deacetylase and sulfite oxidase and deposited as DSM accession number 15716 Acidic endoglucanase or acid cellulase, aspartyl protease, polycopper oxidase, serine-secreted from a strain of Alicyclobacillus sp. Ruboxyl protease, serine protease or HtrA-like serine protease, disulfide isomerase, γ-D-glutamyl-L-diamino acid endopeptidase, endo-β-N-acetylglucosaminidase, peptidyl-prolyl-isomerase, acid phosphatase or phytase or phospholipase C A nucleotide sequence encoding a mature enzyme selected from polysaccharide deacetylase or xylan deacetylase and sulfite oxidase and at least 50%, in particular at least 65%, more particularly at least 70%, more particularly at least 80%, more At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most specific It can comprise a nucleotide sequence having 100% identity, preferably consisting.

Sequence number 1 :
In a particular embodiment, the polynucleotide of the invention encodes an acidic endoglucanase or an acidic cellulase and is at least 70%, more particularly at least 80% more than the nucleotide sequence of positions 73-2877 of SEQ ID NO: 1. At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most specific Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 2
In a particular embodiment, the polynucleotide of the invention encodes a glutamate peptidase and has at least 70%, more particularly at least 80%, more particularly at least at least the nucleotide sequence of positions 97-816 of SEQ ID NO: 2. 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly 100% Comprising or consisting of nucleotide sequences having the identity of:

SEQ ID NOs : 3 and 10 :
In a particular embodiment, the polynucleotide of the invention encodes a polycopper oxidase and is at least 70% more particularly with the nucleotide sequence of positions 76-945 of SEQ ID NO: 3 or positions 148-1791 of SEQ ID NO: 10 Is at least 80%, more particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least It comprises or consists of nucleotide sequences with 99%, or most particularly 100% identity.

SEQ ID NOs : 4 and 5 :
In certain embodiments, the polynucleotide of the invention encodes a serine-carboxyl protease and is at least 70% more specific than the nucleotide sequence of positions 568-1878 of SEQ ID NO: 4 or positions 73-1599 of SEQ ID NO: 5 At least 80%, more particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly Comprise or consist of nucleotide sequences having at least 99%, or most particularly 100% identity.

Sequence number 6 :
In a particular embodiment, the polynucleotide of the invention encodes a serine protease or HtrA-like serine protease and is at least 70%, more particularly at least 80%, with the nucleotide sequence of positions 124-1233 of SEQ ID NO: 6. More particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or Most particularly, it comprises or consists of a nucleotide sequence with 100% identity.

Sequence number 7 :
In a particular embodiment, the polynucleotide of the invention encodes a disulfide isomerase and is at least 70%, more particularly at least 80%, more particularly at least at least the nucleotide sequence of positions 91-633 of SEQ ID NO: 7. 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly 100% Comprising or consisting of nucleotide sequences having the identity of:

SEQ ID NO : 8 :
In a particular embodiment, the polynucleotide of the invention encodes γ-D-glutamyl-L-diamino acid endopeptidase and, more particularly, at least 70%, more particularly the nucleotide sequence of positions 88-798 of SEQ ID NO: 8. Is at least 80%, more particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least It comprises or consists of nucleotide sequences with 99%, or most particularly 100% identity.

SEQ ID NO : 9 :
In certain embodiments, the polynucleotide of the invention encodes endo-β-N-acetylglucosaminidase and is at least 70%, more particularly at least 80%, with the nucleotide sequence of positions 79-2304 of SEQ ID NO: 9. More particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or Most particularly, it comprises or consists of a nucleotide sequence with 100% identity.

Sequence number 11 :
In certain embodiments, the polynucleotide of the invention encodes peptidyl-prolyl-isomerase and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 88-735 of SEQ ID NO: 11 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

Sequence number 12 :
In a particular embodiment, the polynucleotide of the invention encodes acid phosphatase or phytase or phospholipase C, and at least 70%, more particularly at least 80%, with the nucleotide sequence of positions 82-1824 of SEQ ID NO: 12. More particularly at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most Specifically, it comprises or consists of a nucleotide sequence having 100% identity.

SEQ ID NOs : 13 and 14 :
In certain embodiments, the polynucleotide of the invention encodes a polysaccharide deacetylase or xylan deacetylase and is at least 70% of the nucleotide sequence of SEQ ID NO: 13 positions 76-750 or SEQ ID NO: 14 positions 64-972. More specifically at least 80%, more specifically at least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more It comprises or consists of a nucleotide sequence having a specific identity of at least 99%, or most particularly 100%.

SEQ ID NO : 15 :
In a particular embodiment, the polynucleotide of the invention encodes sulfite oxidase and has at least 70%, more particularly at least 80%, more particularly at least at least the nucleotide sequence of positions 88-642 of SEQ ID NO: 15. 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly 100% Comprising or consisting of nucleotide sequences having the identity of:

Sequence number 16 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence at positions 64-771 of SEQ ID NO: 16 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 17 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence at positions 73-3390 of SEQ ID NO: 17 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 18 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 124-744 of SEQ ID NO: 18 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 19 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80% more specific with the nucleotide sequence of positions 76-516 of SEQ ID NO: 19 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 20 :
In certain embodiments, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 91-726 of SEQ ID NO: 20 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 21 :
In certain embodiments, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 73-540 of SEQ ID NO: 21 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

Sequence number 22 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence at positions 76-1431 of SEQ ID NO: 22 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 23 :
In certain embodiments, a polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 58-1020 of SEQ ID NO: 23 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 24 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence of positions 88-1023 of SEQ ID NO: 24 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

SEQ ID NO : 25 :
In a particular embodiment, the polynucleotide of the invention encodes a mature functional polypeptide and is at least 70%, more particularly at least 80%, more specific with the nucleotide sequence at positions 85-1197 of SEQ ID NO: 25 At least 90%, more particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99%, or most particularly Comprises or consists of nucleotide sequences having 100% identity.

  The modification of the nucleotide sequence encoding the polypeptide of the present invention has at least one substitution, deletion and / or insertion compared to an amino acid sequence selected from the mature polypeptide encompassed by SEQ ID NOs: 26-50 Required for the synthesis of polypeptides comprising amino acid sequences.

  It will be apparent to those skilled in the art that such modifications can be made to preserve the function of the enzyme, i.e. outside the region that is critical to the function of the enzyme. Accordingly, amino acid residues that are essential for said function are preferably not susceptible to modification, eg substitution. Amino acid residues essential for said function can be identified according to methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (eg, Cunningham and Wells, 1989, Science 244: 1081-1085). The site of substrate-enzyme interaction can also be determined by conformational analysis, as determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (e.g. de Vos et al.,. 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

  Furthermore, the nucleotide sequence encoding the enzyme of the invention does not give rise to another amino acid sequence of the enzyme encoded by the nucleotide sequence, but corresponds to the codon usage of the host organism intended for the production of the enzyme. Can be modified by the introduction of nucleotide substitutions.

  Introduction of mutations into a nucleotide sequence to exchange one nucleotide for another can be accomplished by site-directed mutagenesis using any method known in the art. . Particularly useful is a method using a supercoiled double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. Oligonucleotide primers, each complementary to the opposite strand of the vector, are extended during temperature cycling with Pfu DNA polymerase. Based on the incorporation of the primer, a mutagenized plasmid containing the attached nickel is generated. Following temperature cycling, the product is specific for methylated and hemimethylated DNA to digest the parent DNA template and select for mutation-containing synthesized DNA. Processed by DpnI. Other methods known in the art can also be used. For a general description of nucleotide substitutions, see Ford et al., 1991, Protein Expression and Purification 2: 95-107.

The present invention also comprises a nucleotide sequence that encodes a polypeptide of the present invention and hybridizes with a polynucleotide probe selected from the group consisting of: under high stringency conditions, preferably very high stringency conditions. Also preferably a polynucleotide comprising:
(I) a complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding the mature polypeptide;
(Ii) a complementary strand to a cDNA sequence contained in a nucleotide sequence selected from the group of regions of SEQ ID NOs: 1-25 encoding the mature polypeptide;
(Iii) a fragment of (i) or (ii) above (J. Sambrook, EF Fritsch, and T) that encodes a secreted polypeptide having the function of its corresponding mature polypeptide encompassed by SEQ ID NOs: 26-50 Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

  As will be appreciated, the details and features relating to hybridization of nucleotide sequences will be the same or similar to the hybridization discussed in the section “Polypeptides of the Invention” herein.

  The present invention also provides information about either the amino acid sequence of the polypeptide of the invention or the nucleotide sequence of the polynucleotide of the invention, in particular the polypeptide or polynucleotide sequence of the invention, in electronic or digital form, eg binary code. Or a storage medium suitable for use in electronic, especially digital devices, comprising in other digital codes. The storage medium may suitably be a magnetic or optical disk and an electronic device or a computing device, and the information may in particular be stored on the storage medium in digital form.

Nucleotide structure:
The invention also includes a nucleic acid comprising a nucleotide sequence of the invention operably linked to one or more control sequences that directs expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Concerning the structure.

  The nucleotide sequence encoding the enzyme of the present invention can be manipulated by various means to provide for expression of the enzyme. Prior to insertion of the nucleotide sequence into the vector, manipulation of the nucleotide sequence may be desired or required depending on the expression vector. Techniques for modifying nucleotide sequences using recombinant DNA methods are well known in the art.

  The control sequence may be a suitable promoter sequence, ie a nucleotide sequence that is recognized by the host cell for expression of the nucleotide sequence. The promoter sequence includes transcriptional control sequences that mediate expression of the polypeptide. A promoter can be any nucleotide sequence that exhibits transcriptional activity in a host cell, such as a mutant, truncated and hybrid promoter, and is either extracellular or homologous or heterologous to the host cell. It is obtained from a gene encoding an intracellular polypeptide.

  Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention, particularly in cellular host cells, are the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis (Bacillus subtilis). ) L-bansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), B-cillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens (Bacillus amyloliguefaciens) Promoter obtained from α-amylase gene (amyQ), Bacillus licheniformis spenicillinase gene (penP), Bacillus subtilis xylA and zylB genes and protozoan β-lactamase gene (Villa-Kamaroff, 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), and the tac promoter (De Boer et al., 1983, Proceedings of the National Academy of Science USA 80: 21-25). Additional promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and Sambrook et al., 1989, supra.

  Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells include Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid Stability α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor mieheilipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium A promoter obtained from a gene encoding oxysporam trypsin-like protease (WO96 / 00787), and Aspergillus niger neutral α- Promoter hybrids from genes encoding amylase and Aspergillus oryzae triose phosphate isomerase, and their mutants, and hybrid promoters.

  In yeast hosts, useful promoters are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphorus Obtained from acid dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romunos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, ie a sequence that is recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3 ′ end of the nucleotide sequence encoding the enzyme. Any terminator that is functional in the host cell of choice may be used in the present invention.
Preferred terminators for filamentous fungal host cells are Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase and Fusarium oxysporum trypsin-like protease Obtained from genes.

  Preferred terminators for yeast host cells are derived from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an appropriate leader sequence, ie, an untranslated region of the mRNA that is important for translation by the host cell. A leader sequence is operably linked to the 5 ′ end of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus nidulans triose phosphate isomerase.

  Suitable leaders for yeast host cells are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glycerel Obtained from the gene for aldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

A control sequence is also operably linked to the 3 ′ end of a polyadenylation sequence, ie, a nucleotide sequence, and when transcribed, is recognized by the host cell as a signal to add a polyadenosine residue to the transcribed mRNA. It can also be a sequence. Any polyadenylation sequence that is functional in the host cell of choice is used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nigerans anthranilate synthase, Fusarium oxysporum trypsin-like protease and Aspergillus niger α-glucosidase Obtained from the gene.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

  The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5 'end of the coding sequence of the nucleotide sequence can include a signal peptide coding region that is naturally ligated in alignment with the translation reading frame and the segment of the coding region that naturally encodes the secreted polopeptide. On the other hand, the 5 'end of the coding sequence may comprise a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region whose coding sequence does not naturally contain a signal peptide coding region is required. On the other hand, the foreign signal peptide coding region can simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide. However, any signal peptide coding region that directs the polypeptide expressed in the secretory pathway can be used in the present invention.

  Effective signal peptide coding regions for bacterial host cells include Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus A signal peptide region obtained from genes for Asterothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are described by Sinomen and Palva, 1993, Microbiological Reviews 57: 109-137.

  Efficient signal peptide coding regions for filamentous fungal host cells include Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheisperaginate proteinase, Humicola insolens cellulase and Humicola A signal peptide coat region obtained from the gene for lanuginosal lipase.

  Useful signal peptides for yeast host cells are derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

  The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of the enzyme. The resulting polypeptide is known as a proenzyme or propolypeptide. A propolypeptide is generally inactive and can be converted from a propolypeptide to a mature active polypeptide by catalytic or autocatalytic degradation of the propeptide. The propeptide coding region contains genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic proteinase gene, and miceriopsola thermophilia laccase. (WO95 / 33836).

  When both the signal peptide and propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located next to the amino terminus of the polypeptide and the signal peptide region is next to the amino terminus of the propeptide region. Located in. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the TAKA α-amylase promoter, Aspergillus niger glucoamylase promoter and Aspergillus oryzae glucoamylase promoter can even be used as regulatory sequences.

  The other columns of regulatory sequences are those sequences that allow gene amplification. In eukaryotic systems, they include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein gene amplified with heavy metals. In those cases, the nucleotide sequence encoding the polypeptide is operably linked by a regulatory sequence.

Recombinant expression vector:
The invention also relates to a recombinant expression vector comprising a nucleic acid construct of the invention. The various nucleotides and control sequences described above generate recombinant expression vectors that can include those sites to allow insertion or substitution of the nucleotide sequence encoding the polypeptide at one or more convenient restriction sites. Can be linked together to On the other hand, the nucleotide sequences of the invention can be expressed by inserting said sequence or a nucleic acid construct comprising said sequence into a suitable vector for expression. In creating an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

  A recombinant expression vector can be any vector (eg, a plasmid or virus) that can be conveniently subjected to recombinant DNA methods and can effect the expression of a nucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

  The vector may be an autonomously replicating vector, ie a vector that exists as a chromosomal entity (its replication is independent of chromosomal replication), such as a plasmid, extrachromosomal element, minichromosome or artificial chromosome.

  The vector can include any means for verifying self-replication. On the other hand, when introduced into a filamentous fungal cell, the vector can be a vector that is integrated into the genome and replicated with the chromosome into which it is integrated. In addition, a single vector or plasmid, or a plurality of vectors or plasmids that together contain the total DNA or transposon introduced into the genome of the host cell can be used.

  The vectors of the present invention preferably contain one or more selectable markers that allow easy selection of transformed cells. A selectable marker is a gene and its product provides biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like.

  Examples of bacterial selectable markers are dal genes from Bacillus subtilis or Bacillus lichenilformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells are selected from, but not limited to: amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase) ), HygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase), and their equivalents.

The amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus are preferred for use in Aspergillus cells.
The vectors of the present invention preferably include elements that allow stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell regardless of the cell's genome.

  For integration into the genome of a host cell, the vector is a nucleotide sequence encoding a variant in the vector for integration of the vector into the genome by homologous or non-homologous recombination, or any other element. Depends on. On the other hand, the vector can contain additional nucleotide sequences to direct integration by homologous recombination into the genome of the host cell. The additional nucleotide sequence allows integration of the vector into the host cell genome at the exact location on the chromosome.

  In order to increase the likelihood of integration at the correct location, the integration element is preferably a sufficient number of nucleic acids that exhibit high homology with the corresponding target sequence to increase the likelihood of homologous recombination, e.g., 100-1500. Should include one base pair, preferably 400-1,500 base pairs, and most preferably 800-1,500 base pairs. The integration element can be any sequence that is homologous to the target formulation in the genome of the host cell. Further, the integration element can be a non-coding or coding nucleotide sequence. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

  For autonomous replication, the vector further comprises an origin of replication that allows the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184, which allow replication in E. coli, and pUB110, pE194, pTA1060 and pAMβ1, which allow replication in Bacillus. Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ie the combination of ARS1, ARS4, ARS1 and CEN3, and the combination of ARS4 and CEN6.

  The origin of replication is the origin having a mutation that makes its function thermosensitive in the host cell (see, eg, Ehrlich, 1978, Procedings of the National Academy of Sciences USA 75: 1433).

  One or more copies of the nucleotide sequence of the invention can be inserted into the host cell to enhance the production of the gene product. An increase in the copy number of the nucleotide sequence is obtained by incorporating at least one additional copy of the sequence into the host cell genome or by including a selectable marker gene that can be amplified with the nucleotide sequence, wherein the cell is a selectable marker An amplified copy of the gene is included, so that additional copies of the nucleotide sequence can be selected by culturing the cells in the presence of a suitable selection agent.

  The methods used to link the above elements to construct the recombinant expression vectors of the invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

Recombinant host cell:
The invention also relates to a recombinant host cell comprising a nucleic acid construct of the invention that is conveniently used in the recombinant production of variants. A vector comprising a nucleotide sequence of the present invention is introduced into a host cell such that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above.
The host cell can be a unicellular microorganism, such as a prokaryotic organism, or a unicellular microorganism, a eukaryotic organism.

  Useful single cells include bacterial cells such as the following Gram-positive bacteria Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausch, Bacillus coagillans, Bacillus lautas, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis, or Streptomyces cells such as Streptomyces lividans or Streptomyces murinus, or the following Gram-negative bacteria: E Coli and pseudomonas sp. (But not limited to). In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

Introduction of vectors into bacterial host cells can be accomplished, for example, by competent cells (eg, Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209- 221) using protoplast transformation (see eg Changand Cohen, 1979, Molecular General Genetics 168: 111-115), electroporation (see eg Shigekawa and Dower, 1988, Biotechniques 6: 742-751) Or a junction (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).
The host cell can be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

In preferred embodiments, "fungi" wherein the host cell is a fungal cell, as used herein, refers to Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (Hawksworth, etc.) ., Ainsworth and Bisby's Dictionary of the Fungi, 8 th edition, 1995, CAB International, University Press, Cambridge, is defined by the UK), and Oomycota (Oomycota) (such as Hawksworth., 1995, said, quoted in 171 pages As well as vegetative spores (Hawksworh et al., 1995, supra).

  In a more preferred embodiment, the “yeast” wherein the fungal host cell is a yeast cell, as used herein, belongs to Endomycetals, Basidiomycete yeast, and Blastomycetes. Includes yeast. Since the classification of yeast may change in the future, for the purposes of the present invention, yeast is considered as Biology and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, eds. Soc. App. Bacteriol. Symposium Series. No. 9, 1980).

  In an even more preferred embodiment, the yeast host cell is Candida, Hunsenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia. .

  In a most preferred embodiment, the yeast host cell is Saccharomyces carlsbergensis, Saccharomyses cerevisiae, Saccharomyces diastaticus, Saccharomyces diastaticus, Saccharomyces diastaticus, Saccharomyces diastaticus, Saccharomyces diastaticus, kluyveri), Saccharomyces norbensisi, or Saccharomyces oviformis cells. In another most preferred embodiment, the yeast host cell is Kluyveromyces lactis. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

  In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filiform fungus” includes all filamentous forms of Eumycota and Omycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelium wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Growth proliferation is by fungal expansion and carbon metabolism is absolutely aerobic. In contrast, growth and proliferation by yeasts such as Saccharomyces cerevisiae is by germination of unicellular fronds and carbon metabolism is fermentable.

  In an even more preferred embodiment, the filamentous fungal host cell is Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium (Penicilium), Thielavia, Tolypocladium or Trichoderma species of cells, but not limited to them.

  In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonica, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell comprises Fusarium bacteriodiides, Fusarium clockwellens, Fusarium ceralis, Fusarium kurumorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium Negunji, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambusium, Fusarium sarcochrome, Fusarium solani, Fusarium sporotricioides, Fusarium sulfureum, Fusarium tolurosum, Fusarium tricosaideum It is a cell.

  In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. Nov.) Cell. In a further most preferred embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Misericopsola thermophilia, Neurospora crassa, Penicillium pulprogenum, Thielavia terrestris, Trichoderma haldianum, Trichoderma konigi, Trichoderma longibrakiatum, Trichoderma reesei or Trichoderma viride cell.

  Fungal cells can be transformed by processes including protoplast transformation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable methods for transformation of Aspergillus host cells are described in European Patent No. 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81; 1474-1874. Suitable methods for transforming a fullerium species are described by Malardier et al., 1989, Gene78: 147-156, and WO96 / 00787. In yeast, Becker and Guarente.In Abelson, JN and Simon, MI, editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito, etc. 1983, Journal of Bacteriology 153: 163; and Hinnnen et al., 1978, Proceedings of the National Academy of Sciences USA 75; 1920.

Donor strain:
The present invention also provides a bacterium Alicyclobacillus sp. Deposited under accession number DSM15716 and a composition comprising this microorganism.

Method for preparing enzyme polypeptide:
The present invention also includes (a) culturing a strain comprising a nucleotide sequence encoding the enzyme of the present invention capable of expressing and secreting the enzyme of the present invention, and (b) recovering said enzyme. And a method for producing the enzyme of the present invention. In a particular embodiment, the strain is a wild-type strain, such as Alicyclobacillus sp. DSM15716, and in another embodiment, the strain is a recombinant host cell as described above.

  In those production methods of the invention, the cells are cultured in a nutrient medium suitable for production of the enzyme using methods known in the art. For example, the cells can be shaken flask cultures, small or large scale fermentations in a suitable medium and in laboratory or industrial fermentors performed under conditions that allow the expression and / or isolation of the polypeptide. (Including continuous, batch, fed-batch, or group state fermentation). Culturing is performed using methods known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are either commercially available or can be prepared according to published compositions (eg, in catalogs of the American Type Culture Collection). If the enzyme is secreted into the nutrient medium, the enzyme can be recovered directly from the medium.

  The resulting enzyme can be recovered by methods known in the art. For example, the enzyme can be recovered from the nutrient medium by conventional methods such as, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation or precipitation.

  The polypeptides of the present invention can be obtained by various methods known in the art, such as chromatography (eg, ion exchange, affinity, hydrophobicity, chromatofocusing and size exclusion), electrophoresis methods (eg, isoelectric for separation). Point electrophoresis), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction (but not limited to) (eg, Protein Purification, JC Janson and Lars Ryden, editors, VCH Publlishers , New York, 1989).

  The method of the invention also fuses a gene from the genome of Alicyclobacillus sp. DSM15716 (eg, from a gene library) and a signalless reporter, eg, a gene encoding β-lactamase, through a transposon label. A host cell clone comprising the gene of Alicyclobacillus sp. DSM15716 fused through a transposon tag with a signalless reporter, for example a gene encoding β-lactamase, a medium representing the presence of the reporter, such as an ampicillin-containing medium WO01 based on a sample of Alicyclobacillus sp. DSM15716 by detecting a clone that grows in and secreting said reporter and isolating the gene and polypeptide of Alicyclobacillus sp. DSM15716 contained in the clone Wrapping TAST method of / 77315A1 To.

  A host cell comprising the gene of Alicyclobacillus sp. DSM15716 fused through transposon labeling with a signalless reporter, for example, a gene encoding β-lactamase, is grown in a medium representing the presence of the reporter, such as an ampicillin-containing medium In some cases, only those clones that express and secrete a reporter (eg, β-lactamase) will be detected (eg, will survive).

  However, a reporter is an intact promoter and ribosome binding site (ie, a gene that is expressed by a cell to produce a polypeptide in real life) in which the gene to which the reporter gene is fused can be recognized in the host strain. ), And if the reporter is translated so that the synthesized polypeptide is transported through the cytoplasmic membrane and correctly folded, it will be secreted. Thus, when a fused gene is inserted into a selected host cell, the presence of a reporter is detected (eg, ampicillin resistance), and those clones are alicyclobacillus encoding a secreted functional polypeptide. It will contain the gene from sp. DSM15716.

Transgenic plants:
The invention also relates to a transgenic plant, plant part or plant cell that has been transformed with a nucleotide sequence encoding the enzyme of the invention to express and produce the enzyme. In one embodiment, the plant can be used as a host for the production of enzymes in recoverable amounts. The enzyme can be recovered from the plant or plant part. On the other hand, the plant or plant part containing the recombinant enzyme is used to improve the quality of food or feed, for example to improve nutritional value, taste and flow properties, or to destroy anti-nutritive factors. Can be used. In particular, the plant or plant part expressing the enzyme can be used as an improved starting material for the production of fuel-alcohol or bio-ethanol.

The transgenic plant can be a dicotyledonous plant or a monocotyledonous plant. Examples of monocotyledonous plants are grasses, for example grasses that prefer wetlands (Strawberry genus grass, Gramineae), grasses, such as Boletus japonica, toboshigara, dokumugi, temperate grasses, such as nukabo, and cereals, such as wheat, Oats, rye, barley, rice, sorghum and corn.
Examples of dicotyledonous plants are tobacco, legumes such as Hauchiwa Bean, potato, sugar beet, peas, beans and soybeans, and Brassicaceae (Brassicaceae) such as cauliflower, rapeseed and closely related model organisms Arabidopsis It is Taraiana (Arabidopsis Thaliana).

Examples of plant parts are stems, callus, leaves, roots, fruits, seeds, and tuberous roots. At present, certain plant tissues such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm appear to be plant parts. Furthermore, any plant cell is considered to be a plant part, whatever its tissue origin.
Such plants, plant parts and plant cell progeny are also encompassed within the scope of the present invention.

  Transgenic plants or plant cells that express the enzymes of the present invention can be constructed according to methods known in the art. Briefly, a plant or plant cell incorporates one or more expression structures encoding an enzyme of the present invention into the plant host genome and the resulting modified plant or plant cell is transformed into a transgenic plant or plant cell. It is composed by growing.

  Conveniently, the nucleic acid construct is operatively associated with a nucleotide sequence encoding an enzyme of the invention together with appropriate regulatory sequences required for expression of said nucleotide sequence in a selected plant or plant part. A DNA structure comprising. In addition, the expression structure comprises a selectable marker that is useful for identifying the host cell in which the expression structure is incorporated, and the DNA sequence required for introduction of the structure into the plant in question. (The latter depends on the DNA introduction method used).

  The selection of regulatory sequences, such as promoter and terminator sequences, and optionally signal or transition sequences, is determined based on the desire of when, where and how the enzyme is expressed. For example, the expression of a gene encoding an enzyme of the invention can be structural or inducible, or progressive, stepwise or tissue specific, and the gene product can be a specific tissue or plant part, such as a seed or Can be targeted to leaves. Regulatory sequences are described, for example, by Tague et al., Plant Phys., 86: 506, 1988.

  For constitutive expression, the 35S-CaMV promoter can be used (Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters are storage tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Annu. Rev. Genet. 24: 275-303), or metabolic tissues such as meristems (Ito et al., 1994). , Plant Mol. 24: 863-878), or seed-specific promoters such as glutelin, prolamin, globulin or albumin promoters from rice (Wu et al., Plant and Cell Physiology Vol. 34, No. 39, No .8, pp.885-889 (1998)), Conrad U. et al., Journal of Plant Physiology Vol. 152, No.6, pp.708-711 (1998), from Vicia faba. Of legumin B4 and unknown seed protein gene, Vicia faba promoter, seed oil body protein promoter (Chen et al., Plant and Cell Physiology Vol.39, No.9, pp.935-941 (1998) ), Storage from Brassica napus It can be a protein napA promoter, or any other seed-specific promoter known in the art such as described for example in WO 91/14772.

  Furthermore, promoters are leaf-specific promoters such as rbcs promoters from strains or tomatoes (Kyozuka et al., Plant Physiology Vol.102, No.3, pp.991-1000 (1993)), methyltransferases from cholera virus. Gene promoter (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol.26, No.1, pp.85-93 (1994)), or aldP gene promoter from rice (Kagaya et al., Molecular and General Genetics Vol. .248, No. 6, pp. 668-674 (1995)), or a wound-inducible promoter, such as a potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22, 573-588).

Promoter enhancer elements can be used to achieve higher enzyme expression in plants. For example, the promoter enhancer element can be an intron placed between the promoter and the nucleotide sequence encoding the enzyme of the invention. For example, Xu et al. Discloses the use of the first intron of the rice actin 1 gene actin 1 gene to enhance expression.
The selectable marker gene and any other portion of the expression construct can be selected from those available in the art.

  Nucleic acid constructs are subject to conventional techniques known in the art, such as Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation. It is integrated into the plant genome (Gasser et al., Science, 244, 1293; Potrykus, Bio / tech. 8,535,1990; Shimamoto et al., Natune, 338, 274, 1989).

  Currently, Agrobacterium tumefaciens-mediated gene transfer is the preferred method for generating transgenic dicotyledonous plants (see Hooykas & Schilperoort, 1992, Plant Mol. Biol. 19: 15-38). However, it can also be used to transform monocotyledonous plants, although other transformation methods are generally preferred for those plants. Currently, the preferred method for generating transgenic monocotyledons is embryo callus or growing embryo particle bombardment (micro gold or tungsten particles coated with transforming DNA) (Christou, 1992, Plant J 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio / Technology 10: 667-674). Another method for transformation of monocotyledons is protoplast transformation, as described by Omirulleh B, et al., Plant Molecular Biology Vol. 21, No. 3, pp. 415-428 (1993). Based on.

Following transformation, transformants incorporating the expression construct are selected and regenerated into complete plants according to methods well known in the art.
The present invention also includes (a) culturing a transgenic plant or plant cell comprising a nucleotide sequence encoding an enzyme of the present invention under conditions suitable for production of the enzyme; and (b) It also relates to a method for producing an enzyme of the invention comprising recovering.

Compositions comprising polypeptides and methods for their preparation:
The invention comprises a composition comprising a polypeptide of the invention and preferably an excipient, and a method for preparing such a composition comprising mixing the polypeptide of the invention and an excipient. I will provide a. In particular, the composition comprises at least 2, preferably at least 3, more preferably at least 5, more preferably at least 10, more preferably at least 15, more preferably at least 20 different polypeptides. It consists of Most compositions will contain all the secreted polypeptide when fermenting a sample of Alicyclobacillus sp. DSM15716 or a variant thereof (one or more genes deleted or added). Comprising.

  In certain embodiments, the polypeptides of the invention are the major (polypeptide) component in a composition, eg, a one-component composition. Excipients herein are to be understood as any auxiliaries or compounds used in formulating the composition and include solvents, carriers, stabilizers, and the like.

  The composition further comprises one or more additional enzymes such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, Glucoamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectin degrading enzyme, peptide glutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase Comprising.

  The composition is prepared according to methods known in the art and can exist in the form of a solution or a solid composition. For example, the enzyme composition can be formulated using methods known in the art for formulating polypeptides and / or pharmaceutical products into coated or uncoated granules or microgranules. Thus, the polypeptides of the present invention can be provided in the form of granules, preferably dust-free granules, liquids, particularly stabilized liquids, slurries or protected polypeptides. For certain applications, immobilization of the polypeptide on a solid matrix is preferred.

  The polypeptide contained in the composition is stabilized according to methods known in the art to stabilize the polypeptide in the composition by adding an antioxidant or reducing agent and limiting the oxidation of the polypeptide. Obtain or stabilize by adding a polymer known to be beneficial to the stability of the polypeptide in a solid or liquid composition, such as PVP, PVA, PEG or other suitable polymer Can be done.

In a further aspect, the composition of the present invention comprises a surfactant, and optionally a builder, such as a zeolite, a bleach, such as a percarbonate, a bleach enhancer, such as TAED, in addition to the polypeptide of the present invention. Or a detergent composition comprising a compound selected from the group consisting of NOBS, soap water suds suppressors, fragrances, and the like.
In a further aspect, the composition of the invention is a feed composition comprising cereals or grains in addition to the polypeptide.

In a further aspect, the composition of the present invention is a food composition, such as a crumb composition, brewed product, fruit juice, oil or lard product, comprising the polypeptide of the present invention.
In a further aspect, the composition of the invention comprises a polysaccharide, or a mixture of polysaccharides, and comprises a polypeptide of the invention.
In a further aspect, the composition of the invention is a pulp composition comprising pulp in addition to the polypeptide of the invention.
In a further aspect, the composition of the invention is a biocidal composition comprising an oxidoreductase enhancer in addition to the polypeptide of the invention.

Use of polypeptides or compositions comprising them:
In a further aspect, the present invention relates to various uses of a polypeptide or polynucleotide of the invention, or a composition comprising said polypeptide or polynucleotide, in particular for (technical) processes, eg for commercial research purposes. For use in industrial or household processes. Accordingly, the present invention includes a method comprising using a polypeptide of the present invention or a polynucleotide of the present invention for (technical) industrial research or household processes.

In one embodiment, the polypeptide or composition of the invention is used for washing cellulose fibers.
In another aspect, the polypeptides or compositions of the invention are used to prepare food or feed additives.
In yet another aspect, the polypeptides or compositions of the invention are used for the treatment of lignol material and pulp.

Detergent disclosure:
The polypeptides of the present invention can be added to a detergent composition and thus become a component of the composition.
The detergent compositions of the present invention may be formulated as manual or machine laundry detergent compositions, such as laundry additive compositions and rinse cloth softener compositions suitable for pretreatment of dyed fabrics, or It can be formulated as a detergent composition for use in general household hard surface cleaning operations, or it can be formulated for manual or machine dishwashing operations.

In a particular aspect, the present invention provides a detergent additive comprising the polypeptide of the present invention. The detergent additive and detergent composition may comprise one or more other enzymes such as proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases such as laccases and / or poloases. It may comprise a xidase.
In general, the nature of the selected enzyme should be compatible with the selected detergent (ie pH-optimal compatibility with other enzymes and non-enzymatic components, etc.) and the enzyme is effective Should be present in quantity.

  Proteases: Suitable proteases include those of animal, plant or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered variants are included. The protease can be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, in particular those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (eg of porcine or bovine origin) and the Fusarium protease described in WO89 / 06270 and WO94 / 25583.

Examples of useful proteases are variants described in WO92 / 19729, WO98 / 20115, WO98 / 20116 and WO98 / 34946, in particular variants having substitutions at one or more of the following positions: : 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
Preferred commercially available protease ^{enzymes, Alcalase TM, Savinase TM, Primase} TM, Duralase TM, Esperase TM and ^{Kannase TM (Novo Nordisk A / S} ), Maxatase TM, Maxacal TM, Maxapem TM, Properase TM, Purafect TM, Purafect OxP TM , FN2 ^™ and FN3 ^™ (Genencor International Inc.).

  Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered variants are included. Examples of useful lipases are Humicola (also known as Thermomyces), for example Humicola Lauginosa (T. Lauginosa) as described in European Patent Nos. 258068 and 305216, or Humicola as described in WO96 / 13580.・ Insolens, P. Alkalinenes or P. pseudoalkaligenes (European Patent No. 218272), P. Sephacia (European Patent No. 331376), P. Stutzeri (UK Patent No. 1,372,034), P. Full Olesense, pseudomonas sp. SD705 strain (WO95 / 06720 and WO96 / 27002), lipase from P. Wisconsinensis (WO96 / 12012), for example, B. subtilis (Dartois et al. (1993) , Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (Japanese Patent 64/744992) or B. pumilas (WO91 / 16422).

Other examples are lipase variants such as WO92 / 05249, WO94 / 01541, European Patent No. 407225, European Patent No. 206105, WO95 / 35381, WO96 / 00292, WO95 / 30744, WO94 / 25578. No., WO95 / 14783, WO95 / 22615, WO97 / 04079 and WO97 / 07202.
Preferred commercially available lipase enzymes include Lipolase ^™ and Lipolase Ultra ^™ (Novo Nordisk A / S).

  Amylase: Suitable amylases (α- and / or β) include those of bacterial or fungal origin. Chemically modified or protein engineered variants are included. Amylases include Bacillus, for example α-amylase obtained from the patent strain of B. lichenylformis, which is described in more detail in British Patent 1,296,839.

Examples of useful amylases are the variants described in WO94 / 02597, WO94 / 18314, WO96 / 23873 and WO97 / 43424, in particular variants having substitutions at one or more of the following positions: : 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408 and 444.
Commercially available amylases are Duramyl ^™ , Termamayl ^™ , Fungamyl ^™ and BAM ^™ (Novo Nordisk A / S), Rapidase ^™ and Purastar ^™ (Genencor International Inc.).

  Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered variants are included. Suitable cellulases are cellulases from the genera Bacillus, Humicola, Fusarium, Thielavia, Acremonium, e.g. U.S. Pat.No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat.No. 5,691,178, U.S. Pat.No. 5,776,757 and WO 89/09259. Including the disclosed Humicola insolens, fungal cellulases produced from Myseriopsola thermophila / oxysporum.

  Particularly suitable cellulases are alkali or neutral cellulases with color protection benefits. Examples of such cellulases are those described in European Patent No. 0495257, European Patent No. 0531372, WO96 / 11262, WO96 / 29397, WO98 / 08940. Examples of examples are WO94 / 07998, European Patent No. 053315, US Patent No. 5,457,046, US Patent No. 5,685,593, US Patent No. 5,763,254, WO95 / 24471, WO98 / 12307 and PCT / DK98 / 0299. Those described in.

Commercial cellulases include Celluzyme ^™ and Carezyme ^™ (Novo Nordisk A / S), Clazinase ^™ and Puradex HA ^™ (Genencor International Inc.) and KAC-500 (B) ^™ (Kao Corporation).

  Peroxidase / oxidase: Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered variants are included. Examples of useful peroxidases include peroxidases from coprinas, such as Coprinus cinereus, and variants thereof such as those described in WO93 / 24618, WO95 / 10602 and WO98 / 15257.

Commercially available peroxidases include Guardzyme ^™ (Novo Nordisk A / S).
The detergent enzyme can be included in the detergent composition by adding a separate additive comprising one or more enzymes or by adding a combined additive comprising all of those enzymes. The detergent additives of the present invention, i.e. separate or combined additives, can be formulated as granules, liquids, slurries, etc., for example. Preferably the detergent additive formulation is a granulate, in particular a non-dusting granule, a liquid, in particular a stable liquid or slurry.

  Non-dusting granules can be produced as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and optionally coated by methods known in the art. Examples of wax coating materials are poly (ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1000-20,000; ethoxylated nonylphenol having 16-50 ethylene oxide units; 12-20 Ethoxylated fatty alcohols; fatty alcohols, fatty acids; and mono-, di- and triglycerides of fatty acids, having an alcohol with 5 carbon atoms and having 15 to 80 ethylene oxide units. An example of a film-forming coating material suitable for application by fluidized bed techniques is given in British Patent No. 1448591. For example, liquid enzyme preparations are stabilized according to established methods by adding polyols such as propylene glycol, sugars or sugar alcohols, sulfuric acid or boric acid. The protected enzyme can be prepared according to the method disclosed in EP 238,216.

The detergent compositions of the present invention can be present in any convenient form, for example in the form of bars, tablets, powders, granules, pastes or liquids. Liquid detergents are aqueous and typically contain up to 70% water and 0-30% organic solvent or may be non-aqueous.
The detergent composition comprises one or more surfactants which may be nonionic, for example semi-polar and / or anionic and / or cationic and / or zwitterionic. Surfactants are typically present at levels of 0.1-60% by weight.

  When included therein, the detergent is typically from about 1% to about 40% anionic surfactant, such as linear alkyl benzene sulfonate, α-olefin sulfonate, alkyl sulfate (fatty acid sulfate), alcohol ethoxy sulfate, second Alkane sulfonates, alpha-sulfo fatty acid methyl esters, alkyl- or alkenyl succinic acids or soaps will be included.

  When included therein, the surfactant is typically about 0.2% to about 40% nonionic surfactant, such as alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyldimethylamine oxides, ethoxylated fatty acids. It may include monoethanolamide, fatty acid monoethanolamide, polyhydroxyalkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucosamide”).

Detergents are 0-65% detergent builders or complexing agents such as zeolites, diphosphates, triphosphates, phosphonates, carbonates, citrates, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminehexaacetic acid, alkyl- or alkynyl succinic acid, Soluble silicates or layered silicates (eg SKS-6 from Hoechst) can be included.
The detergent comprises one or more polymers. Examples include carboxymethylcellulose, poly (vinyl pyrrolidone), poly (ethylene glycol), poly (vinyl alcohol), poly (vinyl pyridine-N-oxide), poly (vinyl imidazole), polycarboxylate (eg, polyacrylate, malein). Acid / acrylic acid copolymer and lauryl methacrylate / acrylic acid copolymer.

The detergent can comprise a perborate or percarbonate that can be combined with a bleaching system comprising a source of H ₂ O ₂ , such as a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. . On the other hand, the bleaching system can comprise peroxyacids of the amide, imide or sulfone type, for example.
The enzyme of the detergent composition of the present invention comprises conventional stabilizers such as polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives such as aromatic boric acid esters or phenylboric acid derivatives such as 4- It can be stabilized with formylphenylboronic acid and the composition can be formulated as described, for example, in WO92 / 19709 and WO92 / 19708.

Detergents also include other conventional detergent compositions such as fabric conditioners such as clays, foam enhancers, soap foam inhibitors, anticorrosives, soil suspensions, anti-soil re-deposition agents, dyes, fungicides, fluorescent Brightening agents, hydrotropes, haze inhibitors, or fragrances can be included.
In the detergent composition, any enzyme, in particular the enzyme of the invention, is 0.01-100 mg enzyme protein per liter of washing liquid, preferably 0.05-5 mg enzyme protein per liter of washing liquid, especially 0.1-1 mg per liter of washing liquid. It is currently planned that it may be added in an amount corresponding to that enzyme protein.
The enzymes of the present invention can further be incorporated into the detergent formulations disclosed in WO 97/07202 (incorporated herein by reference).

Deposited microorganisms:
The following microorganisms have been deposited by the applicant with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany in accordance with the Budapest Treaty based on the international recognition of the deposit of microorganisms for patent procedures:
30 June 2003: Alicyclobacillus sp. CS81 fever-acidoacid: DSM accession number 15716.

Example 1 : Identification of a functional polypeptide secreted by Alicyclobacillus sp. DSM 15716 : Genomic library construction :
Chromosomal DNA from Alicyclobacillus sp. DSM15716 was prepared by using standard molecular biology techniques (Ausuble et al. 1995 "Current protocols in molecular biology" Publ: John Wiley and sons). The prepared DNA was partially cut with Sau3A and separated on an agarose gel. The 3-8 kb fragment was lysed and precipitated and resuspended in the appropriate buffer.

Genomic libraries were produced using the Stratagene ZAP Express predigested Vector kit and Stratagene ZAP Express ^™ predigested Gigapack ™ cloning kit (Bam HI predigested) (Stratagene Inc., USA) according to their vendor's instructions / recommendations . The resulting lambda AZP library contained 38000 pfu and 10,000 of them were collected for mass exclusion. The resulting 70000 E. coli colonies were pooled and plasmids were prepared by using the Qiagen Spin Mini prep kit (Qiagen, Germany). About 1 ml of the eluate containing the plasmid DNA was precipitated by centrifugation at 20000 rpm at 4 ° C. with 1 part by volume of sodium acetate (pH 5) and 2 parts by volume of 96% ethanol, washed with 70% v / v ethanol, Dried at room temperature and resuspended in 200 μl TE buffer. The DNA concentration of the plasmid pool DNA of the Alicyclobacillus sp. Genomic library was 5.2 μg / μl.

Transposon composition and preparation :
The basic principle behind the Transposon Assisted Signal Trapping (TAST) method as described in WO01 / 77315A1 is the transposon of a gene encoding β-lactamase without signal and all genes in the selected genome. Fusing through a label. Thus, when a host cell clone comprising a genomic gene fused with a gene encoding β-lactamase without signal through a transposon label is grown in ampicillin-containing medium, it expresses and secretes β-lactamase Only those clones will survive.

  However, β-lactamase is an intact promoter and ribosome binding site that allows the gene to which the β-lactamase gene is fused to be recognized in the host strain (ie, by cells to produce a polypeptide in true life). And the β-lactamase will be translated so that the synthesized polypeptide will be secreted if it is transported through the plasma membrane and correctly folded. Thus, when inserting a fused gene into a selected host cell, those clones that are ampicillin resistant contain a gene that encodes a secreted functional polypeptide.

Usually, when using the TAST method, it is not necessary to express the entire gene. When a gene is labeled with a transposon, expression of the N-terminal portion of the gene as a protein fusion indicates that the gene contains intact transcription, translation and secretion sequences. Thus, expression of the N-terminal part of a gene as a protein fusion is usually considered sufficient to assume the expression and secretion of the entire gene.
It can therefore be concluded that the gene obtained by the TAST method actually codes for a secreted functional polypeptide.

Construction of SigA4 transposon containing β-lactamase reporter gene:
The construction of a transposon containing β-lactamase without signal was performed using standard molecular biology techniques as described in WO01 / 77315A1. Signalless β-lactamase was first PCR amplified from PUC19 using a proofreading polymerase (Pfu Turbo, Stratagene, USA). The resulting PCR fragment contained restriction sites NotI and EcoRI to aid cloning. Plasmid pEntranceposon (Cam ^r ) containing the entrance transposon and antibiotic resistance marker CAT (encoding chloramphenicol resistance in the transposon) was obtained from Finnzymes, OY (Espoo Finland). The plasmid was digested with restriction enzymes NotI and EcoRI, gel purified and ligated with a signal-free β-lactamase-containing fragment. The ligation was used to transform electro-competent DH10B cells, and an E. coli clone containing a recombinant plasmid and no signal β-lactamase was identified by restriction analysis and designated SigA2.

  For transposon preparation, a smaller derivative of SigA2 lacking the bla gene encoding β-lactamase was constructed: the following two oligonucleotide primers SigA2NotU that bind to the start and stop of the bla gene of SigA2 directed outward -P 5'-TCG CGA TCC GTT TTC GCA TTT ATC GTG AAA CGC T-3 '(SEQ ID NO: 51) and SigA2NotD-P 5'- CCG CAA ACG CTG GTG AAA GTA AAA GAT GCT GAA-3' (SEQ ID NO: 52 ). The approximately 3.6 kb growth produced in this PCR reaction was religated and transformed into the appropriate E. coli strain. A 3.6 kb plasmid was isolated from a transformant that grew on LB chloramphenylchol but failed to grow on LB ampicillin. This plasmid carries both BglI sites and lacks the active Bla gene and was named pSig4.

  60 μl of pSigA4 plasmid DNA preparation with a concentration of 0.3 μg / μl was digested with BglII and separated on an agarose gel. The 2 kb SigA2 transposon DNA band was eluted and purified according to the manufacturer's instructions by using the “GFXPCR, DNA and Gel Band Purification Kit” (Amersham Pharmacia Biotech Inc, USA) and placed in 200 μl EB buffer. Eluted.

C. Transposon labeling:
The transposon prepared from pSigA4 carries a 5'-cleaved bla-gene encoding β-lactamase from which the secretion signal has been removed. β-lactamase transmits ampicillin resistance on E. coli only when the protein is secreted around the cell, whereas cytoplasmic expression of β-lactamase does not confer ampicillin resistance. In the absence of a signal sequence, β-lactamase will not be transported around the cell and thus the clone will not grow on ampicillin containing media.

  The signalless β-lactamase gene was included in the transposon in such a way that there was a continuous reading frame between the transposon boundary and the β-lactamase coding region. In this case, the modified transposon causes a consistent fusion with the target gene when it translocates into the gene encoding the secreted protein. This resulted in a fusion gene product that was secreted around the cells of E. coli and transmitted resistance to ampicillin. If the transposon is integrated into a gene encoding a protein that is not secreted, each host will not be ampicillin resistant.

  For in vitro transposon targeting of the Alicyclobacillus sp. Library, 4 or 8 μl of SigA2 transposon containing approximately 2.6 μg of DNA, 2 μl of plasmid pool DNA of Alicyclobacillus sp. Genomic library at a DNA concentration of 1 μl Mix with 50 μl combined volume with Finnzymes MuA Transposase (0.22 μg / μl) and 5 μl 5 × buffer (from Finnzymes OY, Espoo, Finland) and incubate at 30 ° C. for 3.5 hours and then 75 Heat inactivated at 10 ° C. for 10 minutes. DNA was precipitated by the addition of 5 μl of 3M potassium acetate (pH 5) and 110 μl of 96% ethanol and centrifuged at 20000 rpm for 30 minutes. The pellet was washed and dried and resuspended in 10 μl TE buffer.

D. Transformation and selection:
Electro-competent E. coli DH10B cells were transformed by electroporation in a Biorad Gene Pulse device (50 μF, 25 mAmp, 1.8 kV) with 5 μl of transposon labeled plasmid pool, mixed with 1 ml of SOC medium, 37 Preincubated for 1 hour at 0 ° C. and plated on LB containing 25 μl / ml ampicillin, 50 μl / ml kanamycin, 10 μl / ml chloramphenicol and incubated for 2-3 days. 1056 colonies from resistant transformants were selected and plasmids were prepared by applying the Qiaprep 96 Turbo Bioroboto kit according to the vendor's instructions.

E. Plasmid preparation and resistance determination:
The 1056 transposon-labeled plasmid was downstream in the first reaction by the A2up primer AGCGTTTGCGGCCGCGATCC (SEQ ID NO: 53) which reads the transposon-labeled gene upstream, and in the second reaction the transposon-labeled gene is downstream. Sequencing was performed with B primer TTATTCGGTCGAAAAGGATCC (SEQ ID NO: 54).

F. Sequence assembly and annotations:
The resulting sequence was transformed into the program PhredPhrap (Brent Ewing, LaDeana Hillier, Michael C. Wendl, and Phil Green, Base-calling of automated sequencer traces using phred 1. Accuracy assessment (1998) Genome Research 8: 175-185; Brent Ewing and Phil Green, Base-calling of automated sequencer traces using phred 11. Error probabilities (1998) Genome Research 8: 186-194) and assembled into a contig.

Subsequently, the resulting contig was transferred to the program BLASTX 2.0a19MP-WashU [14-Jul-1998] [Build linux-x86 18: 51: 44 30- Jul-1998] (Gish, Warren (1994-1997). Unpublished; Gish , Warren and David J. States (1993) .Identification of protein coding regions by database similarity search.Nat Genet. 3: 266-72). Compared to.
The resulting sequence was a functional gene encoding an intact and functional polypeptide as they were obtained as ampicillin resistant colonies as previously described.

Example 2 : Determination of function by homology :
The function of polypeptide SEQ ID NO: 26-50 was annotated by sequence comparison with a gene or polypeptide of known function. The polypeptides of the present invention were compared to a list of closely related sequences from Contig's published and internal database. Contigs from which SEQ ID NOS: 26-50 were derived were subsequently compared to sequences available from standard public DNA and protein sequence databases by using the program BLASTX 2 Oa19MP-WashU [14-Jul-1998]. Careful analysis of those closely related sequences with known functions from other databases of sequence alignments SEQ ID NOs: 26-40 allows prediction of the function of those polypeptides based on the degree of amino acid identity I made it.

  Carefully analyze and interpret amino acid residues in the catalytic or critical regions of polypeptide sequences, even when overall amino acid identity is 40% or less, which usually makes good prediction difficult Thus, the functions of SEQ ID NOs: 26 to 40 could be predicted. If an amino acid at the catalytic site of a known sequence is also present in the polypeptide of the invention, the polypeptide from Alicyclobacillus sp. DSM15716 has the same function as the known sequence when combined with sufficient overall amino acid identity. Was concluded.

Example 3 : Preparation of polypeptides of SEQ ID NOs : 26-50 :
In order to prepare the polypeptides of SEQ ID NOs: 26-50, the genes encompassed by SEQ ID NOs: 1-25 encoding those polypeptides are transferred to a suitable host strain, such as E. coli, Bacillus subtilis, Bacillus licheniformis. Alternatively, it is expressed by fusing a DNA encoding an open reading frame to DNA of a promoter, a ribosome binding site and a terminator suitable for gene expression in a derivative of Bacillus claudius or Alicyclobacillus sp. The promoter can be either an inducible promoter or a constitutive promoter. Any signal sequence of SEQ ID NOs: 26-50 can be exchanged for an appropriate signal peptide from other bacteria.

  The expression construct can be part of a plasmid or linear DNA. It can be integrated into the chromosome of the host strain by recombination or it can be present on a plasmid in the host cell. The transformed cells carrying the gene of interest are then grown in a suitable medium in the desired volume. If an inducible promoter is used, the gene begins expression by adding an inducer. On the other hand, no inducer is required and the cells are grown until the appropriate amount of protein from the gene of interest is produced. The culture is then harvested and the protein is recovered by standard methods.

Example 4 : Determination of serine-carboxyl protease activity :
The culture fluid or cell lysate of the host strain that synthesizes and secretes the serine-carboxyprotease in a suitable buffer can be assayed for its activity. An appropriate volume of such sample is spotted on an agarose gel containing the insoluble chromogenic substrate AZCL-collagen (Megazyme ^™ ) or Azocoll (Sigma-Aldrich) and an appropriate buffer at an acidic pH of 3-5. . The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 1 day. The activity appears as a blue circle around the spot. As an alternative to AZCL-collagen or Azocoll, unlabeled collagen can be added onto an agar plate, on which enzyme activity can be detected as a clear area.

  Addition of pepstatin cannot inhibit the protease activity of serine carboxyl protease. As an alternative, the activity determination of samples containing serine-carboxyl protease can be performed by Tsuruoka N, Nakayama T, Ashida M, Hemmi H, Nakao M, Minakata H, Oyama H, Oda K, Nishino T; "Colla- genolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: purifica- tion, characterization, gene cloning, and heterologous expression. "Appl Environ Microbiol. Vol. 69 (1); pp 162-169; 2003 May be measured as described in Jan. .

Example 5 : Determination of multi-copper oxidase activity :
Culture fluids or cell lysates of host strains that synthesize and secrete multi-copper oxidase in an appropriate buffer are described in Schneider et al., Enzyme and Microbial Technology 25, (1999) p. 502-508. Thus, it can be assayed for its activity.
For example, an appropriate volume of such a sample, which may be 15 μl, is transferred to a suitable buffer, eg, 0.1 M sodium acetate buffer (pH 5.5) at a suitable concentration, eg, 1 mM, ABTS (2, 2′− Spot on agarose containing azinobis-3-ethylbenzthiazoline-6-sulfonic acid). The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 16 hours. The activity appears as a green area around the sample. Assays are performed on supernatants and extracts.

Example 6 : Determination of serine protease activity :
Culture fluids or cell lysates of host strains that synthesize and secrete serine proteases in an appropriate buffer can be assayed for their activity. An appropriate volume of such sample is spotted on an agarose gel containing the insoluble chromogenic substrate AZCL-casein (Megazyme ^™ ) or AZCL-collagen (Megazyme ^™ ) and an appropriate buffer at the appropriate pH. The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 1 day. The activity appears as a blue circle around the spot. As an alternative to AZCL-casein or AZCL-collagen (Megazyme ^™ ), unlabeled casein or unlabeled collagen can be used. A transparent region forms in the presence of serine proteases on unlabeled collagen or unlabeled casein spotted on an agarose plate.

Example 7 : Determination of glutamate peptidase activity :
The culture fluid or cell lysate of the host strain that synthesizes and secretes glutamate peptidase in an appropriate buffer can be assayed for its activity. An appropriate volume of such sample is spotted on an agarose gel containing the insoluble chromogenic substrate AZCL-collagen (Megazyme ^™ ) and an appropriate buffer at an acidic pH of 3-5. The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 1 day. The activity appears as a blue circle around the spot.

As an alternative to AZCL-casein, unlabeled collagen can be used. A transparent region forms in the presence of glutamate peptidase on unlabeled collagen spotted on an agarose plate. Based on a specific test of the glutamate peptidase of SEQ ID NO: 27, the activity was determined using 20 μl of culture fluid on 0.1% AZCL-collagen (Megazyme ^™ ) spotted on LB-PG agar plates at pH 3.4. Determined as a spot test. The plate was incubated at 55 ° C. (overnight) and the presence of glutamate peptidase appeared as a blue circle around the spot.

  The glutamate peptidase encompassed by SEQ ID NO: 27 showed significant sequence similarity to peptidases belonging to family A4 currently classified as peptidase family G1 (PepG) (EC3.4.23.19) by MEROPE (SEQ ID NO: 27 , And Fujinaga M, Cherney MM, Oyama H, Oda K, James MN .; The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum; Proc. Natl. Acad. Sci. USA; 101 ( 10); pp. 3364-9; Epub 01-Mar-2004; see 09-Mar-2004). This family includes peptidase sequences with Q and E conserved in their active site. Both residues were conserved in the glutamate peptidase encompassed by SEQ ID NO: 27. Thus, the glutamate peptidase encompassed by SEQ ID NO: 27 is the first bacterial polypeptide of the G1 family that only considers fungal peptidases.

  SEQ ID NO: 27 was compared to a reference sequence of the family G1 peptidase; Aspergillus niger Aspergillopepsin II (SEQ ID NO: 55; Swisssprot P24665; Takahashi, K .; Inoue, H .; Sakai, K .; Kohama, T .; Kitahara, S .; Takishima, K .; Tanji, M .; Athauda, SBP; Takahashi, T .; Akanuma, H .; Mamiya, G .; Yamasaki, M); The primary structure of Aspergillus niger acid proteinase A.; J. Biol. Chem .; Vol 266; p. 19480; 1991). This polypeptide contained a signal peptide (aa1 to aa18) and two propeptides (aa19-58 and aa99-109) that were removed after secretion during maturation. During maturation, H and L chains are formed that are bridged by disulfide bridges between cysteine residues.

  (Inoue, H .; Kimura, T .; Makabe, O .; Takahashi, K .; The gene and deduced protein sequences of the zymogene of Aspergillus niger acid proteinase A; J. Biol. Chem .; vol. 266; p. 19484; 1991). An amino acid similar to the second propeptide (aa99-109) and the amino acid corresponding to the bridging cysteine residue of SEQ ID NO: 55 is deleted in SEQ ID NO: 27 (see single row alignment). Only fungal G1 peptidase has been previously described to lack a cysteine residue (Maita, T .; Nagata, S .; Matsuda, G .; Maruta, S .; Oda, K .; Murao, S .; Tsuru, D .; Complete amino acid sequence of Scytalidium lignicolum acid protease B; J. Biochem .; vol. 95; p. 465;

Single alignment of SEQ ID NO: 27 and SEQ ID NO: 55:

* = Amino acid that forms the active site in Swissprot P24665: = Cysteine residue that forms a disulfide bond in Swissprot P24665
# = Propeptide removed from Swissprot P24665 zymogen.

Example 8 : Determination of acid β-glucanase activity :
A culture fluid or cell lysate of the host strain that synthesizes and secretes acidic β-glucanase in a suitable buffer can be assayed for its activity. An appropriate volume of such sample is spotted on an agarose gel containing the insoluble chromogenic substrate AZCL-β-glucan (Megazyme ^™ ) and an appropriate buffer at an acidic pH of 3-5. The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 1 day. The activity appears as a blue circle around the spot.

Example 9 : Determination of acid phosphatase activity :
To measure enzyme activity in an appropriate volume of culture fluid or cell lysate of a host strain that synthesizes and secretes acid phosphatase in an appropriate buffer at an appropriate temperature, eg, 55 ° C., and at an appropriate pH. Were incubated with para-nitrophenol phosphate (pNPP). The product of the enzymatic reaction is p-nitrophenol and inorganic phosphate or PI. NaOH is added, after an appropriate reaction time, the phosphatase assay is stopped and p-nitrophenolate is formed. The absorbance of p-nitrophenolate is measured optically at 405 nm. Alternatively, an appropriate volume of culture fluid or cell lysate of the host strain that synthesizes and secretes acid phosphatase in an appropriate buffer at an appropriate temperature, eg, 55 ° C., and at an appropriate pH, may be Is used with EnzChek Acid Phosphatase Assay Kit (E-12020) (Molecular Probes Europe BV; Poort Gebouw, Rijnsburger-weg 10; 2333 AA Leiden, The Netherlands).

Example 10 : Determination of polysaccharide deacetylase activity :
An appropriate volume of culture fluid or cell lysate of the host strain that synthesizes and secretes the polysaccharide deacetylase in an appropriate buffer at an appropriate temperature, eg, 55 ° C., was used to measure the activity. Bacterial murein, N, N′-diacetylchitobiose (Sigma) or galactose pentaacetate (Sigma) and / or cellulose acetate (Sigma) can be used as a substrate for this enzyme type. Acetate released from the substrate by the enzyme can be measured with an acetic acid assay kit (Biopharm) adapted for the physical requirements of the enzyme (Kosugi A, Murashima K, and Doi RH; Xylanase and Acetyl Xylan Esterase Vol. 68; pp. 6399-6402; 2002), Activities of XynA, a Key Subunit of the Clostridium cellulovorans Cellulosome for Xylan Degradation; Appl. Environm. I Microbiol.

Example 11 : Determination of endo-β-N-acetylglucosaminidase activity :
An appropriate volume of culture fluid or cell lysate of the host strain that synthesizes and secretes endo-β-N-acetylglucosaminidase in an appropriate buffer (pH 3-5) at an appropriate temperature, eg 55 ° C. , MH Rashid, M Mori and J Sekiguchi; Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization; Microbiology; vol. 141; pp. 2391-2404; 1995, used to measure activity .

Example 12 : Determination of peptidylproyl-isomerase activity :
An appropriate volume of culture fluid or cell lysate of the host strain that synthesizes and secretes peptidylproyl-isomerase in an appropriate buffer at an appropriate temperature, eg 55 ° C., is used to measure the activity. did. Activity of Fischer, G., Bang, H. and Mech, C .; Determination of functional catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides .; Biomed. Biochim. Acta; vol. 43; pp 1101- 1111; can be determined according to 1984. This assay can be suitably modified to match the particular peptidyl-isomerase encompassed by SEQ ID NO: 36.

Example 13 : Determination of acid cellulase activity :
Culture fluids or cell lysates of host strains that synthesize and secrete acid cellulase in an appropriate buffer can be assayed for their activity. An appropriate volume of such sample is spotted on an agarose gel containing the insoluble chromogenic substrate AZCL-HE-cellulose (Megazyme ^™ ) and an appropriate buffer at an acidic pH, eg 3-5. The plate is incubated at an appropriate temperature, eg, 55 ° C., for an appropriate time, eg, 1 day. The activity appears as a blue circle around the spot.

Example 14 : Determination of xylan deacetylase activity :
An appropriate volume of culture fluid or cell lysate of the host strain that synthesizes and secretes the polysaccharide deacetylase in an appropriate buffer at an appropriate temperature, eg, 55 ° C., was used to measure the activity. Xylan deacetylase activity was prepared from birchoxylan by the method of Johnson et al. 1988 (Johnson, KG, JD Fontana, and CR Mackenzie. 1988. Measurement of acetylxylan esterase in Streptomyces. Methods Enzymol. 160: 551-560). Measured as acetate released from acetylated xylan.

  The acetate released from acetyl xylan can be measured with an acetic acid assay kit (Biopharm) adapted for the physical requirements of the enzyme (Kosugi A, Murashima K, and Doi RH; Xylanase and Acetyl Xylan Esterase Activities vol. 68; pp. 6399-6402; 2002) of XynA, a Key Subunit of the Clostridium cellulovorans Cellulosome for Xylan Degradation; Appl. Environm. I Microbiol.

Example 15 : Measurement of phytase activity :
A culture fluid or cell lysate of the host strain that synthesizes and secretes phytase in an appropriate buffer can be assayed for its phytase activity. An appropriate volume of such a sample is prepared with HCl at pH 3.0-3.5, sodium acetate at pH 4.0-5.5, morpholine ethane sulfonic acid (MES) at pH 6.0-6.5 and pH 7.0-9.0. Diluted with 0.1 M sodium acetate and 0.01% Tween-20 (pH 5.5) in a suitable buffer, which can be Tris-HCl, and further diluted with substrate solution (0.1 M sodium acetate and 0.01% Tween-20 ( Dilute 26-fold in 5 mM sodium phytate (Sigma) in pH 5.5) and preincubate at 37 ° C. to initiate the reaction. After 30 minutes at 37 ° C., the reaction is stopped by adding an equal volume of 10% trichloroacetic acid.

Free inorganic phosphate was measured by addition of an equal volume of molybdate reagent containing 7.3 g FeSO ₄ , 1.0 g (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O and 3.2 ml H ₂ SO ₄ in 100 ml To do. Absorbance was measured at 750 nm (Vmax microtiter plate reader; Molecular Devices) (Lassen SF; Breinholt J; Ostergaard PR; Brugger R; Bischoff A; Wyss M; Fuglsang CC; Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora Iycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens; Appl. Environ. Micr .; 67; pp. 4701-4707; 2001).

Example 16 : Measurement of phospholipase activity :
Culture fluids or cell lysates of host strains that synthesize and secrete phospholipases in an appropriate buffer can be assayed for their phospholipase activity. Lecithin is added to an appropriate volume of such sample. Lecithin is hydrolyzed at a constant pH and temperature, and phospholipase activity is determined as the rate of titrant (0.1 N NaOH) digestion during neutralization of the liberated fatty acid. The substrate is soy lecithin (L-α-phosphotidyl-choline) and the conditions are pH 8.00, 40.0 ° C., 2 minutes reaction time. Isolation (LEU) is defined relative to a standard.

Example 17 : Expression of glutamate peptidase gene (SEQ ID NO : 2) in Bacillus subtilis :
A signal peptide from the protease SAVINNASE ^™ (also known as subtilisin 309 from B. licheniformis from Novozymes A / S) is read by PCR into the gene encoding glutamate peptidase (SEQ ID NO: 2) The frames were aligned and fused. The DNA encoding the resulting coding sequence was integrated into the Bacillus subtilis host cell genome by homologous recombination.

  The gene structure consists of a promoter from Bacillus thuringiensis cryIII A containing the Bacillus licheniformis α-amylase gene (amyL), Bacillus amyloliquefaciens α-amylase gene (amyQ), and a stabilizing sequence. It was expressed under the control of a triple promoter system (as described in WO99 / 43835). The gene encoding chloramphenicol acetyltransferase was used as a marker. (Diderichsen et al., A useful cloning vector for Bacillus subtilis. Plasmid, 30, p. 312, 1993).

Chloramphenicol resistant transformants were analyzed by DNA sequencing to confirm the correct DNA sequence of the construct. One such clone was selected.
Fermentation of glutamate peptidase (SEQ ID NO: 2) expressing clones was performed on a rotary shake table in 500 ml baffled flasks each containing 100 ml PS-1 medium supplemented with 6 mg / l chloramphenicol. Clones were fermented at 37 ° C. for 6 days, and samples were taken on days 3, 4, 5 and 6 and analyzed for proteolytic activity. Activity was determined as a spot test of 20 μl culture fluid on 0.1% AZCL-collagen (Megazyme ^™ ) LB-PG agar plates at pH 3.4 (see Example 7). Plates were incubated at 55 ° C. (overnight) and activity appeared as a blue circle around the spot.

Example 18 : Purification and characterization of family A4 protease from Alicyclobacillus sp.
Purification :
The culture broth was centrifuged (20000 × g, 20 minutes) and the supernatant was carefully decanted from the precipitate. The combined supernatant was filtered through a Seitz EKS plate to remove the remainder of the Bacillus host cells. The EKS filtrate was adjusted to pH 4.0 with citric acid and heated to 70 ° C. with stirring on a water bath. When the solution reached 70 ° C. (it took about 15 minutes from 25 ° C. to 70 ° C.), the solution was immediately placed on ice. This heat treatment results in some precipitation, which is removed by another Seitz EKS filter plate filtration. Ammonium sulfate is added to the second EKS filtrate to a final concentration of 1.6 M and the plate is equilibrated in 20 mM CH ₃ COOH / NaOH, 1.6 M (NH ₄ ) ₂ SO ₄ (pH 4.5). Applied to Butyl Toyopearl S column. After intensive washing of the Butyl column with equilibration buffer, the enzyme was eluted with a linear (NH ₄ ) ₂ SO ₄ gradient (1.6-0M) in the same buffer.

Fractions from the column were analyzed for protease activity (using assay buffer at pH 4.0 and assay temperature of 36 ° C.) and the active fractions were pooled. Pooled fractions were transferred to 20 mM CH ₃ COOH / NaOH (pH 5.5) on a G25 Sephadex column and applied to a SOURCE 30Q column equilibrated with the same buffer. After intensive washing of the SOURCG 30Q column with equilibration buffer, the protease was eluted with a linear NaCl gradient (0-0.5M) in the same buffer.

  Fractions from the column were analyzed for protease activity (pH 4.0, 37 ° C.) and active fractions were pooled. The slightly colored pool was treated with 1% (w / v) activated charcoal for 5 minutes and the charcoal was removed by 0.45 μm filtration. The purity of the filtrate was analyzed by SDS-PAGE, where only one band was seen on the Coomassie stained gel.

Assay :
Pratazyme OL (cross-linked and stained collagen) assay was used. Protazyme OL tablets (from Megazyme) were suspended in 2.0 ml of 0.01% Triton X-100 with gentle agitation. 500 μl of this suspension and 500 μl of assay buffer were mixed in an Epandorf tube and placed on ice. 20 μl protease sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Epandorf tube to an Epandorf thermomixer set at the assay temperature.

The tube was incubated for 15 minutes on an Eppendorf thermomixer at its highest shaking speed (1400 rpm). Incubation was stopped by transferring the tube to an ice bath. The tubes were then centrifuged for several minutes in an ice-cooled centrifuge, 200 μl of the supernatant was transferred to a microtiter plate, and the OD ₆₅₀ was read at 650 nm. A buffer blind was included in the assay (instead of the enzyme). OD ₆₅₀ (enzyme) -OD ₆₅₀ (buffer blind) was a measure of protease activity.

Protease assay:
Substrate: Protazyme OL tablets (Megazyme T-PROL).
Temperature: adjusted.
Assay buffer: pH-value 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, with 100 mM oxalic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl ₂ , 150 mM KCI, HCI or NaOH 0.01% Triton X-100 adjusted to 8.0, 9.0, 10.0, 11.0 and 12.0.

Characterization: pH-activity, pH-stability and temperature-activity :
The protease assay was used to obtain a pH activity profile, a pH-stability profile and a temperature-activity profile at pH 3.0. For the pH stability profile, the protease was diluted 5-fold with assay buffer and incubated at 37 ° C. for 2 hours. Following incubation, the protease samples were transferred to pH 3.0 by dilution with pH 3 assay buffer prior to assaying for residual activity.

Other features :
The relative molecular weight of A4 protease as determined by SDS-PAGE was Mr = 26 kDa.

Example 19 : Expression of an acid cellulase gene (SEQ ID NO : 1) in Bacillus subtilis :
A signal peptide from Termanyl ^™ (Novozymes) was fused with acid cellulase to the coding-sul gene (SEQ ID NO: 1) by matching the open reading frame by PCR. The DNA encoding the resulting coding sequence was integrated into the Bacillus subtilis host cell genome by homologous recombination. The gene structure is composed of three promoters consisting of a Bacillus licheniformis α-amylase gene (amyL), a Bacillus amyloliquefaciens α-amylase gene (amyQ) and a promoter from the Bacillus swangiensis cryIII A promoter containing a stabilizing sequence. It was expressed under the control of the original promoter system (as described in WO99 / 43835). A gene encoding chloramphenicol acetyl-transferase was used as a marker (described in Diderichsen et al., A useful cloning vector for Bacillus subtilis. Plasmid, 30, p. 312, 1993).

  Chloramphenicol resistant transformants were analyzed by DNA sequencing to confirm the correct DNA sequence of the construct. One such clone was selected.

Acidic cellulase (SEQ ID NO: 1) expressing clones were fermented on a rotary shake table in 500 ml baffled flasks each containing 100 ml PS-1 medium supplemented with 6 mg / l chloramphenicol. Clones were fermented at 37 ° C. for 3 days and samples were taken on days 1, 2 and 3 and analyzed for cellulase activity. Activity was determined as a spot test of 20 μl culture fluid on 0.1% AZCL-HE-cellulase (Megazyme ^™ ) LB-PG agar plates at pH 3.4. Plates were incubated at 55 ° C. (overnight) and activity appeared as a blue circle around the spot.

Claims (14)

  A polypeptide having glutamic acid peptidase or family 4 protease activity and comprising an amino acid sequence of the first amino acid Leu to the 240th amino acid Ser shown in SEQ ID NO: 27.   A polypeptide having an amino acid sequence having glutamic acid peptidase or family 4 protease activity and having at least 90% amino acid sequence identity to the amino acid sequence of the first amino acid Leu to the 240th amino acid Ser shown in SEQ ID NO: 27 . A polynucleotide encoding the polypeptide according to claim 1 or 2 . An expression vector comprising the polynucleotide according to claim 3 . A host cell transformed with the expression vector according to claim 4 . A method for producing the polypeptide according to claim 1 or 2 , comprising a step of culturing the host cell according to claim 5 . A composition comprising one or more polypeptides according to claim 1 or 2 . 8. The composition of claim 7 , further comprising one or more additional enzymes. The composition according to claim 7 or 8 , which is a detergent composition further comprising a surfactant. The composition according to claim 7 or 8 , which is a food composition. 11. The food composition according to claim 10 , further comprising a polysaccharide or a mixture of polysaccharides. The composition according to claim 7 or 8 , which is a feed composition. 13. A feed composition according to claim 12 , further comprising cereal or grain.   Bacterial strain deposited under the deposit number DSM 15716.